New amidino derivatives and their use as thrombin inhibitors

ABSTRACT

There is provided compounds of formula I,  
                 
 
     wherein R 1 , R x , Y, R y , n and B have meanings given in the description which are useful as competitive inhibitors of trypsin-like proteases, such as thrombin, and in particular in the treatment of conditions where inhibition of thrombin is required (e.g. thrombosis) or as anticoagulants.

FIELD OF THE INVENTION

[0001] This invention relates to novel pharmaceutically usefulcompounds, in particular competitive inhibitors of trypsin-like serineproteases, especially thrombin, their use as medicaments, pharmaceuticalcompositions containing them and synthetic routes to their production.

BACKGROUND

[0002] Blood coagulation is the key process involved in both haemostasis(i.e. the prevention of blood loss from a damaged vessel) and thrombosis(i.e. the formation of a blood clot in a blood vessel, sometimes leadingto vessel obstruction).

[0003] Coagulation is the result of a complex series of enzymaticreactions. One of the ultimate steps in this series of reactions is theconversion of the proenzyme prothrombin to the active enzyme thrombin.

[0004] Thrombin is known to play a central role in coagulation. Itactivates platelets, leading to platelet aggregation, convertsfibrinogen into fibrin monomers, which polymerise spontaneously intofibrin polymers, and activates factor XIII, which in turn crosslinks thepolymers to form insoluble fibrin. Furthermore, thrombin activatesfactor V and factor VIII leading to a “positive feedback” generation ofthrombin from prothrombin.

[0005] By inhibiting the aggregation of platelets and the formation andcrosslinking of fibrin, effective inhibitors of thrombin would beexpected to exhibit antithrombotic activity. In addition, antithromboticactivity would be expected to be enhanced by effective inhibition of thepositive feedback mechanism.

PRIOR ART

[0006] The early development of low molecular weight inhibitors ofthrombin has been described by Claesson in Blood Coagul. Fibrinol.(1994) 5, 411.

[0007] Blombäck et al (in J. Clin. Lab. Invest. 24, suppl. 107, 59,(1969)) reported thrombin inhibitors based on the amino acid sequencesituated around the cleavage site for the fibrinogen Aα chain. Of theamino acid sequences discussed, these authors suggested the tripeptidesequence Phe-Val-Arg (P9-P2-P1, hereinafter referred to as the P3-P2-P1sequence) would be the most effective inhibitor.

[0008] Thrombin inhibitors based on dipeptidyl derivatives with anα,ω-aminoalkyl guanidine in the P1-position are known from U.S. Pat. No.4,346,078 and International Patent Application WO 93/11152. Similar,structurally related, dipeptidyl derivatives have also been reported.For example International Patent Application WO 94/29336 disclosescompounds with, for example, aminomethyl benzamidines, cyclic aminoalkylamidines and cyclic aminoalkyl guanidines in the P1-position; EuropeanPatent Application 0 648 780, discloses compounds with, for example,cyclic aminoalkyl guanidines in the P1-position.

[0009] Thrombin inhibitors based on peptidyl derivatives, also havingcyclic aminoalkyl guanidines (e.g. either 3- or4-aminomethyl-1-amidinopiperidine) in the P1-position are known fromEuropean Patent Applications 0 468 231, 0 559 046 and 0 641 779.

[0010] Thrombin inhibitors based on tripeptidyl derivatives witharginine aldehyde in the P1-position were first disclosed in EuropeanPatent Application 0 185 390.

[0011] More recently, arginine aldehyde-based peptidyl derivatives,modified in the P3-position, have been reported. For example,International Patent Application WO 93/18060 discloses hydroxy acids,European Patent Application 0 526 877 des-amino acids, and EuropeanPatent Application 0 542 525 O-methyl mandelic acids in the P3-position.

[0012] Inhibitors of serine proteases (e.g. thrombin) based onelectrophilic ketones in the P1-position are also known. For example,European Patent Application 0 195 212 discloses peptidyl α-keto estersand amides, European Patent Application 0 362 002 fluoroalkylamideketones, European Patent Application 0 364 344 α,β,δ-triketocompounds,and European Patent Application 0 530 167 α-alkoxy ketone derivatives ofarginine in the P1-position.

[0013] Other, structurally different, inhibitors of trypsin-like serineproteases based on C-terminal boronic acid derivatives of arginine andisothiouronium analogues thereof are known from European PatentApplication 0 293 881.

[0014] More recently, thrombin inhibitors based on peptidyl derivativeshave been disclosed in European Patent Application 0 669 317 andInternational Patent Applications WO 95/35309, WO 95/23609 and WO96/25426.

[0015] However, there remains a need for effective inhibitors oftrypsin-like serine proteases, such as thrombin. There is a particularneed for compounds which are both orally bioavailable and selective ininhibiting thrombin over other serine proteases. Compounds which exhibitcompetitive inhibitory activity towards thrombin would be expected to beespecially useful as anticoagulants and therefore in the therapeutictreatment of thrombosis and related disorders.

DISCLOSURE OF THE INVENTION

[0016] According to the invention there is provided a compound offormula I,

[0017] wherein

[0018] R¹ represents H, C₁₋₄ alkyl (optionally substituted by one ormore substituents selected from cyano, halo, OH, C(O)OR^(1a) orC(O)N(R^(1b))R^(1c)) or OR^(1d);

[0019] R^(1d) represents H, C(O)R¹¹, SiR¹²R¹³R¹⁴ or C₁₋₆ alkyl, whichlatter group is optionally substituted or terminated by one or moresubstituent selected from OR¹⁵ or (CH₂)_(q)R¹⁶;

[0020] R¹², R¹³ and R¹⁴ independently represent H, phenyl or C₁₋₆ alkyl;

[0021] R¹⁶ represents C₁₋₄ alkyl, phenyl, OH, C(O)OR¹⁷ or C(O)N(H)R¹⁸;

[0022] R¹⁸ represents H, C₁₋₄ alkyl or CH₂C(O)OR¹⁹;

[0023] R¹⁵ and R¹⁷ independently represent H, C₁₋₆ alkyl or C₁₋₃alkylphenyl;

[0024] R^(1a), R^(1b), R^(1c), R¹¹ and R¹⁹ independently represent H orC₁₋₄ alkyl; and

[0025] q represents 0, 1 or 2;

[0026] R_(x) represents a structural fragment of formula IIa, IIb orIIc,

[0027] wherein

[0028] the dotted lines independently represent optional bonds;

[0029] A and B independently represent O or S, CH or CH₂ (asappropriate), or N or N(R²¹) (as appropriate);

[0030] D represents —CH₂—, O, S, N(R²²), —(CH₂)₂—, —CH═CH—, —CH₂N(R²²)—,—N(R²²)CH₂—, —CH═N—, —N═CH—, —CH₂O—, —OCH₂—, —CH₂S— or —SCH₂—;

[0031] X₁ represents C₂₋₄ alkylene; C₂₋₃ alkylene interrupted by Z;—C(O)-Z-A¹; -Z-C(O)-A¹-; —CH₂—(O)-A¹; -Z-C(O)-Z-A²-; —CH₂-Z-C(O)-A²-;-Z-CH₂-C(O)-A²-; -Z-CH₂-S(O)_(m)-A²-; —CH₂-Z-S(O)_(m)-A²-; —C(O)-A³;-Z-A³-; or -A³-Z-;

[0032] X₂ represents C₂₋₃ alkylene, —C(O)-A⁴- or -A⁴-C(O)—;

[0033] X₃ represents CH or N;

[0034] X₄ represents a single bond, O, S, C(O), N(R²³), —CH(R²³)—,—CH(R²³)—CH(R²⁴)— or —C(R²³)═C(R²⁴)—;

[0035] A¹ represents a single bond or C₁₋₂ alkylene;

[0036] A² represents a single bond or —CH₂—,

[0037] A³ represents C₁₋₃ alkylene;

[0038] A⁴ represents C(O) or C₁₋₂ alkylene;

[0039] Z represents, at each occurrence, O, S(O)_(m) or N(R²⁵);

[0040] m represents, at each occurrence, 0, 1 or 2;

[0041] R² and R⁴ independently represent one or more optionalsubstituents selected from C₁₋₄ alkyl (which latter group is optionallysubstituted by one or more halo substituent), C₁₋₄ alkoxy,methylenedioxy, halo, hydroxy, cyano, nitro, SO₂NH₂, C(O)OR²⁶ orN(R²⁷)R²⁸);

[0042] R³ represents an optional substituent selected from OH or C₁₋₄alkoxy;

[0043] R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷ and R²⁸ independently representH or C₁₋₄ alkyl;

[0044] Y represents CH₂, (CH₂)₂, CH═CH, (CH₂)₃, CH₂CH═CH or CH═CHCH₂,which latter three groups are optionally substituted by C₁₋₄ alkyl,methylene, oxo or hydroxy;

[0045] R^(y) represents H or C₁₋₄ alkyl;

[0046] n represents 0, 1, 2, 3 or 4; and

[0047] B represents a structural fragment of formula IIIa, IIIb or IIIc

[0048] wherein

[0049] X⁵, X⁶, X⁷ and X⁸ independently represent CH, N or N—O;

[0050] X⁹ and X¹⁰ independently represent a single bond or CH₂; and

[0051] R³¹ represents an optional substituent selected from halo andC₁₋₄ alkyl;

[0052] or a pharmaceutically acceptable salt thereof;

[0053] provided that:

[0054] (a) A and B do not both represent O or S;

[0055] (b) B and D do not both represent O or S;

[0056] (c) when R¹ represents OR^(1d) and X₁ represents —C(O)-Z-A¹,-Z-CH₂-S(O)_(m)-A²-, —CH₂-Z-S(O)_(m)-A²- or -Z-C(O)-Z-A², then A¹ or A²(as appropriate) do not represent a single bond; and

[0057] (d) when X₄ represents —CH(R²³)—, R¹ does not represent OH.

[0058] The compounds of formula I may exhibit tautomerism. Alltautomeric forms and mixtures thereof are included within the scope ofthe invention. Further it will be appreciated by those skilled in theart that, in the structural fragment of formula IIa, the optional doublebonds, may, in conjunction with certain identities of substituent D,render the ring bearing A, B and D aromatic in character.

[0059] The compounds of formula I may also contain one or moreasymmetric carbon atoms and may therefore exhibit optical and/ordiastereoisomerism. All diastereoisomers may be separated usingconventional techniques, e.g. chromatography or fractionalcrystallisation. The various stereoisomers may be isolated by separationof a racemic or other mixture of the compounds using conventional, e.g.fractional crystallisation or HPLC, techniques. Alternatively thedesired optical isomers may be made by reaction of the appropriateoptically active starting materials under conditions which will notcause racemisation or epimerisation, or by derivatisation, for examplewith a homochiral acid followed by separation of the diastereomericderivatives by conventional means (e.g. HPLC, chromatography oversilica). All stereoisomers are included within the scope of theinvention.

[0060] Alkyl groups which R¹, R^(1a), R^(1b), R^(1c), R^(1d), R², R⁴,R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²¹, R²², R²³, R²⁴, R²⁵,R²⁶, R²⁷, R²⁸, R³¹ and R^(y) may represent, and with which Y may besubstituted; the alkyl part of alkylphenyl groups which R¹⁵ and R¹⁷ mayrepresent; and alkoxy groups which R², R³ and R⁴ may represent, may,when there is a sufficient number of carbon atoms, be linear orbranched, saturated or unsaturated, cyclic or acyclic. Alkylene groupswhich X₁, X₂, A¹, A³ and A⁴ may represent may, when there is asufficient number of carbon atoms, be linear or branched, saturated orunsaturated.

[0061] Halo groups, which R³¹ may represent, and with which R¹, R² andR⁴ may be substituted, include fluoro, chloro, bromo and iodo.

[0062] In the structural fragments of formulae IIa, IIb and IIc, thedots indicate the carbon atom which is bonded to the —C(O)— group and toR¹ in a compound of formula I (for the avoidance of doubt, there is nofurther H atom bonded to the carbon atom so indicated).

[0063] The wavy lines on the bond in the fragments of formulae IIIa,IIIb and IIIc signify the bond position of the fragment.

[0064] According to a further aspect of the invention there is provideda compound of formula I as hereinbefore defined with the additionalprovisos that:

[0065] R^(y) represents H;

[0066] R²⁸ represents H;

[0067] X₄ does not represent —CH(R²³)—.

[0068] According to a further aspect of the invention there is provideda compound of formula I as hereinbefore defined with the additionalprovisos that:

[0069] R^(y) represents C₁₋₄ alkyl;

[0070] R²⁸ represents C₁₋₄ alkyl;

[0071] X₄ represents —CH(R²³)—.

[0072] Abbreviations are listed at the end of this specification.

[0073] When n represents 2 and B represents a structural fragment offormula IIIb, preferred compounds of formula I include those wherein X⁹and X¹⁰ do not both represent CH₂.

[0074] Preferred compounds of formula I include those wherein:

[0075] R¹ represents OH or C₁₋₄ alkyl (which latter group is optionallysubstituted by cyano or OH);

[0076] R_(x) represents a structural fragment of formula IIa;

[0077] when R_(x) represents a structural fragment of formula IIa, thedotted lines represent bonds, A and B both represent CH and D represents—CH═CH—;

[0078] when R_(x) represents a structural fragment of formula IIa, X₁represents C₂- or C₃-alkylene, —OCH₂— or —O(CH₂)₂—;

[0079] Y represents CH₂, (CH₂)₂ or (CH₂)₃;

[0080] B represents a structural fragment of formula IIIa in which X⁵,X⁶, X⁷ and X⁸ all represents CH.

[0081] More preferred compounds of the invention include those wherein,when R_(x) represents a structural fragment of formula IIa, X₁represents C₃-alkylene or —O(CH₂)₂—.

[0082] When R_(x) represents a structural fragment of formula IIa, andR² represents at least one substituent, a preferred point ofsubstitution is at the carbon atom which is at position B.

[0083] When R_(x) represents a structural fragment of formula IIa, thedotted lines represent bonds, A and B both represent CH and D represents—CH═CH— (i.e. the ring bearing R² is a benzo group), and R² representsat least one substituent, the ring is preferably substituted either atthe carbon atom in the —CH═CH— group (position D) which is adjacent tothe ring junction, or, more preferably, at the carbon atom which is atposition B, or at both of these sites. For example, when the fragmentIIa represents a tetralin-1-yl group (i.e. the dotted lines representbonds, A and B both represent CH, D represents —CH═CH— and X₁ representssaturated C₃-alkylene), preferred substitution positions are at the 5-or, especially, at the 7-position, or at both to of these positions.Correspondingly, when the fragment IIa represents a chroman-4-yl group(i.e. the dotted lines represent bonds, A and B both represent CH, Drepresents —CH═CH— and X₁ represents —O(CH₂)₂—), preferred substitutionpositions are at the 8- or, especially, at the 6-position, or at both ofthese positions.

[0084] Compounds of formula I in which the fragment

[0085] is in the S-configuration are preferred. The wavy lines on thebonds in the above fragment signify the bond position of the fragment.

[0086] Preferred compounds of formula I include the compounds of theExamples described hereinafter.

PREPARATION

[0087] According to the invention there is also provided a process forthe preparation of compounds of formula I which comprises:

[0088] (i) the coupling of a compound of formula IV,

[0089] wherein R¹ and R_(x) are as hereinbefore defined with a compoundof formula V,

[0090] wherein R^(y), Y, n and B are as hereinbefore defined; or

[0091] (ii) the coupling of a compound of formula VI,

[0092] wherein R¹, R_(x), and Y are as hereinbefore defined with acompound of formula VII,

H(R^(y))N—(CH₂)_(n)—B  VII

[0093] wherein R^(y), n and B are as hereinbefore defined,

[0094] for example in the presence of a coupling agent (e.g. oxalylchloride in DMF, EDC, DCC, HBTU, HATU or TBTU), an appropriate base(e.g. pyridine, 2,4,6,-trimethylpyridine, DMAP, TEA or DIPEA) and asuitable organic solvent (e.g. dichloromethane, acetonitrile or DMF).

[0095] Compounds of formula IV are commercially available, are wellknown in the literature, or are available using known and/or standardtechniques.

[0096] For example, compounds of formula IV in which R¹ represents OHmay be prepared by reaction of a compound of formula VIII,

R_(x)═O  VIII

[0097] wherein R_(x) is as hereinbefore defined, with:

[0098] (a) KCN, for example at 20° C. in the presence of sodiumbisulphite in water, followed by hydrolysis in the presence of aqueousacid (e.g. HCl), for example at 20° C. in the presence of a suitablesolvent (e.g. alcohol and/or water);

[0099] (b) CHCl₃, in the presence of aqueous base (e.g. NaOH);

[0100] (c) TMSCN, for example at 20° C. in the presence of a suitableorganic solvent (e.g. CH₂Cl₂), followed by hydrolysis in the presence ofacid (e.g. HCl or H₂SO₄), for example at 20° C. (e.g. according, oranalogously, to the method described by Bigge et al in J. Med. Chem.(1993) 36, 1977), followed by alkaline hydrolysis to give the free acid.

[0101] Compounds of formula IV in which R¹ represents H may be preparedfrom corresponding compounds of formula IV in which R¹ represents OH (ora lower alkyl ester of the acid), for example by elimination of water,followed by hydrogenation of the resultant alkene using techniques whichare well known to those skilled in the art, followed by, if necessary,hydrolysis to give the free acid.

[0102] Compounds of formula IV in which R¹ represents C₁₋₄ alkyl may beprepared from corresponding compounds of formula IV in which R¹represents H (or a lower alkyl ester of the acid), for example byreaction with an appropriate alkyl halide using techniques which arewell known to those skilled in the art, followed by, if necessary,hydrolysis to give the free acid.

[0103] Compounds of formula IV in which R¹ represents OR^(1d) and R^(1d)represents C(O)R¹¹, SiR¹²R¹³R¹⁴ or C₁₋₆ alkyl may be prepared byacylation, silylation or alkylation (as appropriate) of a correspondingcompound of formula IV in which R¹ represents OH (or a lower alkyl esterof the acid) under conditions which are well known to those skilled inthe art, followed by, if necessary, hydrolysis to give the free acid.

[0104] Compounds of formula V may be prepared by reaction of a compoundof formula IX

[0105] wherein Y is as hereinbefore defined with a compound of formulaVII as hereinbefore defined, for example under conditions such as thosedescribed hereinbefore for synthesis of compounds of formula I.

[0106] Compounds of formulae V and VII in which R^(y) represents C₁₋₄alkyl may be prepared by reaction of a corresponding compound of formulaV or formula VII, as appropriate, in which R^(y) represents H with acompound of formula IXa,

R^(y)Hal  IXa

[0107] wherein Hal represents halo (e.g. Cl, Br or I) and R^(y) is ashereinbefore defined, for example under conditions which are well knownto those skilled in the art.

[0108] Compounds of formula VI are readily available using knowntechniques. For example, compounds of formula VI may be prepared byreaction of a compound of formula IV as hereinbefore defined with acompound of formula IX as hereinbefore defined, for example underconditions such as those described hereinbefore for synthesis ofcompounds of formula I.

[0109] Compounds of formula VIII are commercially available, are wellknown in the literature, or may be prepared in accordance with knowntechniques. For example compounds of formula VIII may be prepared asfollows:

[0110] (a) Compounds of formula VIII in which R_(x) represents astructural fragment of formula IIa, in which the dotted lines representbonds, A and B both represent CH and D represents —CH═CH—; X₁ representsC₂₋₄ alkylene, -Z-A³- or —C(O)-A³-, in which A³ is as hereinbeforedefined; and R³ is absent, may be prepared by cyclisation of a compoundof formula X,

[0111] wherein X_(1a) represents C₂₋₄ alkylene, -Z-A³- or —C(O)-A³-, andZ, A³ and R² are as hereinbefore defined, using an appropriate acylatingagent. for example at 100° C. in the presence of polyphosphoric acid orusing PCl₅ at reflux followed by AlCl₃. Compounds of formula X in whichX_(1a) represents C₃-alkylene or —C(O)-A³-, in which A³ representsC₂-alkylene, may be prepared in accordance with known techniques, forexample by reaction of succinic anhydride with the corresponding phenyllithium and, for compounds of formula X in which X_(1a) representsC₃-alkylene, selective reduction of the resultant ketone, underconditions which are well known to those skilled in the art. Compoundsof formula X in which X_(1a) represents -Z-A³- and A³ represents C₂₋₃alkylene may be prepared as described hereinafter.

[0112] (b) Compounds of formula VIII in which R_(x) represents astructural fragment of formula IIa, in which the dotted lines representbonds, A and B both represent CH and D represents —CH═CH—; X₁ representsC₂₋₄ alkylene or —C(O)-A³-, in which A³ is as hereinbefore defined; andR³ is absent, may alternatively be prepared by cyclisation of a compoundof formula XI,

[0113] wherein R represents C₁₋₆ alkyl and X_(1a) and R² are ashereinbefore defined, for example at 20° C. in the presence of asuitable base (e.g. an alkali metal alkoxide) and an appropriate organicsolvent (e.g. lower alkyl alcohol) followed by hydrolysis anddecarboxylation. Compounds of formula XI may be prepared in accordancewith known techniques. For example, compounds of formula XI in whichX_(1a) represents C₃-alkylene or —C(O)-A³- in which A³ representsC₂-alkylene may be prepared by reaction of succinic anhydride with acompound of formula XII,

[0114] wherein R¹ represents C₁₋₆ alkyl and R and R² are as hereinbeforedefined and, for compounds of formula XI in which X_(1a) representsC₃-alkylene, selective reduction of the resultant ketone, followed byfunctional group transformations of the amide and the acid to estergroups, under conditions which are well known to those skilled in theart.

[0115] (c) Compounds of formula VIII in which R_(x) represents astructural fragment of formula IIa, in which the dotted lines representbonds, A and B both represent CH and D represents —CH═CH—; X₁ represents-Z-A³- in which A³ represents C₂ alkylene and Z represents O or S; andR³ is absent, may be prepared by cyclisation of a compound of formulaXIII,

[0116] wherein Hal and R² are as hereinbefore defined, for example at20° C. in the presence of aqueous-ethanolic NaOH. For correspondingcompounds of formula VIII in which X₁ represents -Z-A³- and Z representsS(O)_(m) in which m is 1 or 2, this abovementioned cyclisation should befollowed by carrying out an oxidation reaction on the cyclised productcomprising an S atom, for example using m-chloroperbenzoic acid.

[0117] (d) Compounds of formula VIII in which R_(x) represents astructural fragment of formula IIa, in which the dotted lines representbonds, A and B both represent CH and D represents —CH═CH—; X₁ represents-Z-A³- in which A³ represents C₂-alkylene or -Z-C(O)-A¹ in which A¹represents C₁-alkylene; and R³ is absent, may be prepared by reaction ofa compound of formula XIV,

[0118] wherein R² and Z are as hereinbefore defined, with either:

[0119] (1) for compounds of formula VIII in which X₁ represents -Z-A³-in which A³ represents C₂-alkylene, a compound of formula XV,

H₂C═CH—CO₂R  XV

[0120] wherein R is as hereinbefore defined, for example at 20° C. inthe presence of a suitable base (e.g. triethylamine or sodium ethoxide)and an appropriate organic solvent (e.g. ethanol or DMF); or

[0121] (2) a compound of formula XVI,

L¹-G-CH₂—CO₂R  XVI

[0122] wherein L¹ represents a suitable leaving group (such as Cl, Br,I, mesylate or tosylate), G represents CH₂ or C(O) and R is ashereinbefore defined, for example at 20° C. in the presence of asuitable base (e.g. triethylamine) and an appropriate organic solvent(e.g. THF); followed by cyclisation under appropriate conditions (e.g.those described hereinbefore).

[0123] (e) Compounds of formula VIII in which R represents a structuralfragment of formula IIa, in which the ring bearing A, B and D is acarbocyclic aromatic, or heterocyclic aromatic, ring as definedhereinbefore in respect of compounds of formula I; X₁ represents—CH₂-Z-C₁₋₂ alkylene-, in which Z is as hereinbefore defined; and R³ isabsent, may be prepared by reaction of a compound of formula XVII,

[0124]  

[0125] wherein the ring bearing A^(a), B^(a) and D^(a) is a carbocyclicaromatic, or heterocyclic aromatic, ring as defined hereinbefore inrespect of compounds of formula I, and Z and R² are as hereinbeforedefined, with a compound of formula XVIII,

L¹-Alk-CO₂H  XVIII

[0126] wherein Alk represents C₁₋₂ alkylene and L¹ is as hereinbeforedefined, for example at 20° C. in the presence of a suitable base (e.g.sodium methoxide) and an appropriate organic solvent (e.g. THF).

[0127] (f) Compounds of formula VIII in which R_(x) represents astructural fragment of formulae IIb, IIc or IIa, in which latter casethe ring bearing A, B and D is a carbocyclic aromatic, or heterocyclicaromatic, ring as defined hereinbefore in respect of compounds offormula I; and, in the cases when R_(x) represents a structural fragmentof formulae IIa or IIb, R³ is absent, may be prepared by cyclisation ofa compound of formula XX,

R_(xa)—CO₂H  XX

[0128] wherein R_(xa) represents a structural fragment of formula XXa,XXb or XXc

[0129] wherein, in XXa, the ring bearing A^(a), B^(a) and D^(a) is acarbocyclic aromatic, or heterocyclic aromatic, ring as definedhereinbefore in respect of compounds of formula I, and R², R⁴, X₁, X₂,X₃ and X₄ are as hereinbefore defined, in the presence of polyphosphoricacid, for example at 100° C. The dots adjacent to the carbon atoms infragments of formula XXa, XXb and XXc signify the point of attachment ofthe fragments to the CO₂H group of the compound of formula XX. Compoundsof formula XX may be prepared by hydrolysis of a corresponding compoundof formula XXI,

R_(xa)—CO₂R  XXI

[0130] wherein R_(ax) and R are as hereinbefore defined (and in whichthe CO₂H in the fragments of formulae XXa, XXb and XXc in R_(xa) mayalso be replaced by CO₂R), for example under reaction conditions whichare well known to those skilled in the art.

[0131] (g) Compounds of formula VIII in which R_(x) represents astructural fragment of formula IIa in which the ring bearing A, B and Dis a carbocyclic aromatic, or heterocyclic aromatic, ring as definedhereinbefore in respect of compounds of formula I; X₁ represents—O—CH₂—; and R³ is absent, may be prepared by reaction of a compound offormula XXII,

[0132] wherein the ring bearing A^(a), B^(a) and D^(a) is a carbocyclicaromatic, or heterocyclic aromatic, ring as defined hereinbefore inrespect of compounds of formula I, and R², Hal and R are as hereinbeforedefined, with diazomethane, for example at 20° C. in the presence of asuitable organic solvent (e.g. diethyl ether).

[0133] (h) Compounds of formula VIII in which R_(x) represents astructural fragment of formula IIa, in which the dotted lines representbonds, A and B both represent CH and D represents —CH═CH—; X₁ represents—C(O)—O—CH₂—; and R³ is absent, may be prepared by cyclisation of acompound of formula XXIII,

[0134] wherein R² and R are as hereinbefore defined, for example at −20°C. in the presence of sulphuric acid and an appropriate organic solvent(e.g. methanol). Compounds of formula XXIII may be prepared by reactinga corresponding acid halide with diazomethane, for example at 20° C. inthe presence of a suitable organic solvent (e.g. diethyl ether).

[0135] (i) Compounds of formula VIII in which R_(x) represents astructural fragment of formula IIa or IIc in which X₁ includes N(R²⁵),or X₄ represent N(R²³), (as appropriate), and R²³ and R²⁵ (asappropriate) represent C₁₋₄ alkyl may be prepared by reaction of acorresponding compound of formula VIII in which X₁ includes, or X₄represents, (as appropriate) NH with a compound of formula XXV

R^(a)-Hal  XXV

[0136] wherein R^(a) represents C₁₋₄ alkyl and Hal is as hereinbeforedefined, for example under conditions which are well known to thoseskilled in the art.

[0137] j) Compounds of formula VIII in which R_(x) represents astructural fragment of formula IIa, in which the dotted lines representbonds, A and B both represent CH and D represents —CH═CH—; X₁ represents—C(O)—N(H)—CH₂—; and R³ is absent, may be prepared by catalytichydrogenation of an hydroxamic acid of formula XXVI,

[0138] wherein R² is as hereinbefore defined, using an appropriatecatalyst system e.g. Pd/C) in the presence of a suitable organic solvent(e.g. methanol). Compounds of formula XXVI may be prepared bycyclisation of a corresponding compound of formula XXVII,

[0139] wherein R² is as hereinbefore defined, for example at 20° C. inthe presence of fuming HCl and tin dichloride.

[0140] (k) Selective oxidation of a compound of formula XXX,

H—R_(x)—H  XXX

[0141] wherein R_(x) is as hereinbefore defined, for example in thepresence of a suitable oxidising agent (e.g. CrO₃ or KMnO₄) and anappropriate solvent (e.g. water).

[0142] (l) Selective oxidation of a compound of formula XXXI,

H—R_(x)—OH  XXXI

[0143] wherein R_(x) is as hereinbefore defined, for example in thepresence of a suitable oxidising agent (e.g. MnO₂) in an appropriateorganic solvent (e.g. CH₂Cl₂).

[0144] (m) Hydrolysis of an oxime formula XXXII,

R_(x)═N—OH  XXXII

[0145] wherein R_(x) is as hereinbefore defined, for example by heatingin the presence of acid (e.g. HCl) and an appropriate organic solvent.Compounds of formula XXXII may be prepared by reaction of acorresponding compound of formula XXX, as hereinbefore defined, withpropyl nitrite, for example in the presence of HCl in ethanol.

[0146] (n) Compounds of formula VIII in which R_(x) represents astructural fragment of formula IIa and X₁ represents —CH₂—CH═CH—, may beprepared by elimination of a compound of formula XXXIII,

[0147] wherein L² represents a suitable leaving group (e.g. Br or SePh)and the dotted lines, A, B, D, R² and R³ are as hereinbefore defined,under appropriate reaction conditions, for example in the presence ofaqueous ethanolic NaOH or hydrogen peroxide, and an appropriate organicsolvent (e.g. THF).

[0148] (o) Compounds of formula VIII in which R_(x) represents astructural fragment of formula IIb, X₂ represents —C(O)-A⁴- and A⁴ is ashereinbefore defined, may be prepared by cyclisation of a compound offormula XXXIV,

[0149] wherein R^(b) represents H, C₁₋₆ alkyl or Hal and R², R³, A⁴, X₃and Hal are as hereinbefore defined, for example in the presence ofpolyphosphoric acid, as described hereinbefore or, in the case whereR^(b) represents Hal, in the presence of AlCl₃ in nitromethane at, forexample, 20° C.

[0150] (p) Compounds of formula VIII in which R_(x) represents astructural fragment of formula IIb and X₂ represents -A⁴-C(O)— and A⁴represents C₁₋₂ alkylene may be prepared by cyclisation of a compound offormula XXXV,

[0151] wherein A^(4a) represents C₁₋₂ alkylene and Hal, R², R³ and X₃are as hereinbefore defined.

[0152] Compounds of formulae VII, IX, IXa, XII, XIII, XIV, XV, XVI,XVII, XVIII, XXI, XXII, XXV, XXVII, XXX, XXXI, XXIII, XXIV and XXV areeither commercially available, are well known in the literature, or areavailable using known techniques, including techniques which are thesame as, or analogous to, those described herein.

[0153] Substituents on the aromatic and/or non-aromatic, carbocyclicand/or heterocyclic ring(s) in compounds of formulae I, IV, V, VI, VII,VIII, X, XI, XII, XIII, XIV, XVII, XX, XXI, XXII, XXIII, XXVI, XXVII,XXX, XXXI, XXXII, XXXIII, XXXIV and XXV may be interconverted usingtechniques well known to those skilled in the art. For example, nitromay be reduced to amino, hydroxy may be alkylated to give alkoxy, alkoxymay be hydrolysed to hydroxy, alkenes may be hydrogenated to alkanes,halo may be hydrogenated to H, etc.

[0154] The compounds of formula I may be isolated from their reactionmixtures using conventional techniques.

[0155] It will be appreciated by those skilled in the art that in theprocess described above the functional groups of intermediate compoundsmay need to be protected by protecting groups.

[0156] Functional groups which it is desirable to protect includehydroxy, amino and carboxylic acid. Suitable protecting groups forhydroxy include trialkylsilyl or diarylalkylsilyl groups (e.g.t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl) andtetrahydropyranyl. Suitable protecting groups for carboxylic acidinclude C₁₋₆ alkyl or benzyl esters. Suitable protecting groups foramino, amidino and guanidino include t-butyloxycarbonyl,benzyloxycarbonyl or 2-trimethylsilylethoxycarbonyl (Teoc). Amidino andguanidino nitrogens may also be protected by hydroxy or alkoxy groups,and may be either mono- or diprotected.

[0157] The protection and deprotection of functional groups may takeplace before or after coupling, or before or after any other reaction inthe abovementioned schemes.

[0158] In particular, the compounds of formula I may be prepared byprocesses comprising the coupling of an N-acylated amino acid or aN-protected amino acid. When a N-protected amino acid is used, the acylgroup may be introduced after coupling. Deprotection of the nitrogenatom may then be effected using standard methods.

[0159] Protecting groups may be removed in accordance with techniqueswhich are well known to those skilled in the art and as describedhereinafter.

[0160] Certain protected derivatives (which may also be referred to as“intermediates”) of compounds of formula I, which may be made prior to afinal deprotection stage to form compounds of formula I, are novel.

[0161] According to a further aspect of the invention there is provideda compound of formula Ia,

[0162] wherein B¹ represents a structural fragment of formula IIId, IIIeor IIIf

[0163] wherein D¹ and D² independently represent H, OH, OR^(a),OC(O)R^(b), OC(O)OR^(c), C(O)OR^(d), C(O)R^(e); in which

[0164] R^(a) represents phenyl, benzyl, C₁₋₇ alkyl (which latter groupis optionally interrupted by oxygen or is optionally substituted byhalo) or —C(R^(f))(R^(g))—OC(O)R^(h);

[0165] R^(b) represents C₁₋₁₇ alkyl (which latter group is optionallysubstituted by C₁₋₆ alkoxy, C₁₋₆ acyloxy, amino or halo); C₁₋₆ alkoxy,C₃₋₇ cycloalkyl, phenyl, naphthyl or C₁₋₃ alkylphenyl (which latter fivegroups are optionally substituted by C₁₋₆ alkyl or halo); or—[C(R^(i))(R^(j))]_(m)OC(O)R^(k);

[0166] R^(c) represents C₁₋₁₇ alkyl, phenyl, 2-naphthyl (which latterthree groups are optionally substituted by C₁₋₆ alkyl,Si(R^(aa))(R^(ab))(R^(ac)) or halo), —[C(R^(m))(R^(n))]_(n)OC(O)R^(p),or —CH₂—Ar¹;

[0167] R^(d) represents 2-naphthyl, phenyl, C₁₋₃ alkylphenyl (whichlatter three groups are optionally substituted by C₁₋₆ alkyl, C₁₋₆alkoxy, nitro, Si(R^(ba))(R^(bb))(R^(bc)) or halo), C₁₋₁₂ alkyl (whichlatter group is optionally substituted by C₁₋₆ alkoxy, C₁₋₆ acyloxy orhalo), —[C(R^(q))(R^(r))]_(p)OC(O)R^(s) or —CH₂—Ar²;

[0168] R^(e) represents phenyl, benzyl, C₁₋₆ alkyl (which latter groupis optionally interrupted by oxygen) or—[C(R^(t))(R^(u))]_(r)OC(O)R^(v);

[0169] R^(aa), R^(ab), R^(ac), R^(ba), R^(bb) and R^(bc) independentlyrepresent C₁₋₆ alkyl or phenyl; R^(f), R^(g), R^(i), R^(j), R^(m),R^(n), R^(q), R^(r), R^(t) and R^(u) independently represent H or C₁₋₆,alkyl;

[0170] R^(h), R^(k), R^(p), R^(s) and R^(v) independently representC₁₋₁₇ alkyl (which latter group is optionally substituted by C₁₋₆alkoxy, C₁₋₆ acyloxy or halo); C₁₋₆ alkoxy, C₃₋₇ cycloalkyl, phenyl,naphthyl or C₁₋₃ alkylphenyl (which latter five groups are optionallysubstituted by C₁₋₆ , alkyl or halo);

[0171] Ar¹ and Ar² independently represent the structural fragment

[0172] m and r independently represent 3 or 4;

[0173] n and p independently represent 1, 2 or 3; and

[0174] R¹, R_(x), Y, R^(y), n, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰ and R³¹ are ashereinbefore defined;

[0175] or a pharmaceutically acceptable salt thereof;

[0176] provided that D¹ and D² do not both represent H.

[0177] Alkyl groups which R^(a), R^(aa), R^(ab), R^(ac), R^(b), R^(ba),R^(bb), R^(bc), R^(c), R^(d), R^(e), R^(f), R^(g), R^(h), R^(i), R^(j),R^(k), R^(m), R^(n), R^(p), R^(q), R^(r), R^(s), R^(t), R^(u) and R^(v)may represent and with which R^(b), R^(c), R^(d), R^(h), R^(k), R^(p),R^(s) and R^(v) may be substituted; cycloalkyl groups which R^(b),R^(h), R^(k), R^(p), R^(s) and R^(v) may represent; the C₁₋₃ alkyl partof alkylphenyl groups which R^(b), R^(d), R^(h), R^(k), R^(p), R^(s) andR^(v) may represent; alkoxy groups which R^(b), R^(h), R^(k), R^(p),R^(s) and R^(v) may represent; and alkoxy and acyloxy groups with whichR^(b), R^(d), R^(h), R^(k), R^(p), R^(s) and R^(v) may be substituted,may, when there is a sufficient number of carbon atoms, be linear orbranched, and may be saturated or unsaturated.

[0178] Halo groups with which R^(a), R^(b), R^(c), R^(d), R^(h), R^(k),R^(p), R^(s) and R^(v) may be substituted include fluoro, chloro, bromoand iodo.

[0179] The wavy lines on the bond in the fragments of formulae IIId,IIIe or IIIf signify the bond position of the fragment.

[0180] Preferred compounds of formula Ia include those wherein D¹represents H and D² represents OH, OCH₃, OC(O)R^(b) or C(O)OR^(d),wherein R^(b) and R^(d) are as hereinbefore defined.

[0181] Compounds of formula Ia may also be prepared directly fromcompounds of formula I in accordance with techniques well known to thoseskilled in the art.

[0182] For example, compounds of formula Ia in which D¹ or D² representsC(O)OR^(d) may be prepared by reaction of a corresponding compound offormula I with a compound of formula XXXVa,

L³-C(O)OR^(d)  XXXVa

[0183] wherein L³ represents a leaving group such as Hal orp-nitrophenoxy, and Hal and R^(d) are as hereinbefore defined forexample at 0° C. in the presence of a suitable base (e.g. NaOH) and anappropriate organic solvent (e.g. THF).

[0184] Compounds of formula Ia may also be prepared directly from othercompounds of formula Ia in accordance with techniques well known tothose skilled in the art.

[0185] Compounds of formula Ia in which D¹ or D² represents OH may beprepared by reaction of a corresponding compound of formula Ia in whichD¹ or D² (as appropriate) represents COOR^(d) and R^(d) is ashereinbefore defined with hydroxylamine (or a hydrohalide salt thereof),for example at 40° C. in the presence of a suitable base (e.g. TEA) andan appropriate organic solvent (e.g. THF).

[0186] Compounds of formula Ia in which D¹ or D² represents OC(O)OR^(c),and R^(c) is as hereinbefore defined, may be prepared by reaction of acorresponding compound of formula Ia in which D¹ or D² (as appropriate)represents OH with a compound of formula XXXVI,

L³C(O)OR^(c)  XXXVI

[0187] wherein L³ and R^(c) are as hereinbefore defined, for example atroom temperature in the presence of a suitable base (e.g. TEA, pyridineor DMAP) and an appropriate organic solvent.

[0188] Compounds of formula Ia in which D¹ or D² represents OC(O)R^(b),and R^(b) is as hereinbefore defined, may be prepared by reaction of acorresponding compound of formula Ia in which D¹ or D² (as appropriate)represents OH with a compound of formula XXXVIa,

R^(b)C(O)L⁴  XXXVIa

[0189] wherein L⁴ represents a suitable leaving group such as OH, Hal orR^(b)C(O)O, and Hal and R^(b) are as hereinbefore defined, for exampleat or below room temperature in the presence of a suitable base (e.g.TEA, pyridine or DMAP) and an appropriate organic solvent (e.g. CH₂Cl₂).

[0190] Compounds of formula Ia in which B¹ represents a structuralfragment of formula IIId (in which X⁵, X⁶, X⁷ and X⁸ all represent CH)or IIIf, in which, in both cases, D¹ represents H and D² represents OHor OR^(a) wherein R^(a) is as hereinbefore defined may alternatively beprepared by reaction of a compound of formula XXXVII,

[0191] wherein B^(a) represents phenyl-1,4-ene or cyclohexyl-1,4-ene andR¹, R_(x), Y, R^(y) and n are as hereinbefore defined with a compound offormula XXXVIII,

H₂NOR^(a1)tm XXXVIII

[0192] wherein R^(a1) represents H or R^(a) and R^(a) is as hereinbeforedefined, for example at between 40 and 60° C., in the presence of asuitable base (e.g. TEA) and an appropriate organic solvent (e.g. THF,CH₃CN, DMF or DMSO). Compounds of formula Ia in which D¹ or D²represents OH or OR^(a) may alternatively be prepared in an analogousfashion by reaction of a corresponding compound of formula Ia, whereinD¹ or D² (as appropriate) represent C(O)OR^(d), and R^(d) is ashereinbefore defined, with a compound of formula XXXVIII, as definedabove.

[0193] Compounds of formula XXXVII may be prepared in accordance withpeptide coupling techniques, for example in analogous fashion to themethods described hereinbefore for compounds of formula I. Compounds offormulae XXXVa, XXXVI, XXXVIa and XXXVIII are commercially available,are well known in the literature, or are available using knowntechniques.

[0194] According to a further aspect of the invention there is provideda compound of formula Ia as defined above except that:

[0195] R^(b) and R^(c) independently represent C₁₋₁₇ alkyl, phenyl or2-naphthyl (all of which are optionally substituted by C₁₋₆ alkyl orhalo);

[0196] R^(d) represents 2-naphthyl, phenyl, C₁₋₃ alkylphenyl (whichlatter three groups are optionally substituted by C₁₋₆ alkyl, C₁₋₆alkoxy, nitro or halogen), CH(R^(f))(CH(R^(g)))_(p)OC(O)R^(h) (in whichR^(f) and R^(g) independently represent H or C₁₋₆ alkyl, R^(h)represents 2-naphthyl, phenyl, C₁₋₆ alkoxy or C₁₋₈ alkyl (which lattergroup is optionally substituted by halo, C₁₋₆ alkoxy or C₁₋₆ acyloxy),and p represents 0 or 1) or C₁₋₁₂ alkyl (which latter group isoptionally substituted by C₁₋₆ alkoxy, C₁₋₆ acyloxy or halogen);

[0197] R^(a) and R^(e) independently represent phenyl, benzyl,(CH₂)₂OC(O)CH₃ or C₁₋₆ alkyl which latter group is optionallyinterrupted by oxygen;

[0198] or a pharmaceutically acceptable salt thereof.

[0199] Persons skilled in the art will appreciate that, in order toobtain compounds of formula I, or formula Ia, in an alternative, and, onsome occasions, more convenient, manner, the individual process stepsmentioned hereinbefore may be performed in a different order, and/or theindividual reactions may be performed at a different stage in theoverall route (i.e. substituents may be added to and/or chemicaltransformations performed upon, different intermediates to thosementioned hereinbefore in conjunction with a particular reaction). Thismay negate, or render necessary, the need for protecting groups.Accordingly, the order and type of chemistry involved will dictate theneed, and type, of protecting groups as well as the sequence foraccomplishing the synthesis.

[0200] The use of protecting groups is fully described in ‘ProtectiveGroups in Organic Chemistry’, edited by J W F McOmie, Plenum Press(1973), and ‘Protective Groups in Organic Synthesis’, 2nd edition, T WGreene & P G M Wutz, Wiley-Interscience (1991).

[0201] The protected derivatives of compounds of formula I (e.g.compounds of formula Ia) may be converted chemically to compounds offormula I using standard deprotection techniques (e.g. hydrogenation),for example as described hereinafter.

[0202] It will also be appreciated by those skilled in the art that,although such protected derivatives of compounds of formula I (e.g.compounds of formula Ia) may not possess pharmacological activity assuch, they may be administered parenterally or orally and thereaftermetabolised in the body to form compounds of formula I which arepharmacologically active. Such derivatives may therefore be described as“prodrugs”. All prodrugs of compounds of formula I are included withinthe scope of the invention.

[0203] Protected derivatives of compounds of formula I which areparticularly useful as prodrugs include compounds of formula Ia.

[0204] Compounds of formula I, pharmaceutically-acceptable salts,tautomers and stereoisomers thereof, as well as prodrugs thereof(including compounds of formula Ia which are prodrugs of compounds offormula I), are hereinafter referred to together as “the compounds ofthe invention”.

Medical and Pharmaceutical Use

[0205] The compounds of the invention are useful because they possesspharmacological activity. They are therefore indicated aspharmaceuticals.

[0206] According to a further aspect of the invention there is thusprovided the compounds of the invention for use as pharmaceuticals.

[0207] In particular, the compounds of the invention are potentinhibitors of thrombin either as such or, in the case of prodrugs, afteradministration, for example as demonstrated in the tests describedbelow.

[0208] The compounds of the invention are thus expected to be useful inthose conditions where inhibition of thrombin is required.

[0209] The compounds of the invention are thus indicated in thetreatment and/or prophylaxis of thrombosis and hypercoagulability inblood and tissues of animals including man.

[0210] It is known that hypercoagulability may lead to thrombo-embolicdiseases. Conditions associated with hypercoagulability andthrombo-embolic diseases which may be mentioned include inherited oracquired activated protein C resistance, such as the factor V-mutation(factor V Leiden), and inherited or acquired deficiencies inantithrombin III, protein C, protein S, heparin cofactor II. Otherconditions known to be associated with hypercoagulability andthrombo-embolic disease include circulating antiphospholipid antibodies(Lupus anticoagulant), homocysteinemi, heparin induced thrombocytopeniaand defects in fibrinolysis. The compounds of the invention are thusindicated both in the therapeutic and/or prophylactic treatment of theseconditions.

[0211] The compounds of the invention are further indicated in thetreatment of conditions where there is an undesirable excess of thrombinwithout signs of hypercoagulability, for example in neurodegenerativediseases such as Alzheimer's disease.

[0212] Particular disease states which may be mentioned include thetherapeutic and/or prophylactic treatment of venous thrombosis andpulmonary embolism, arterial thrombosis (eg in myocardial infarction,unstable angina, thrombosis-based stroke and peripheral arterialthrombosis) and systemic embolism usually from the atrium duringarterial fibrillation or from the left ventricle after transmuralmyocardial infarction.

[0213] Moreover, the compounds of the invention are expected to haveutility in prophylaxis of re-occlusion (ie thrombosis) afterthrombolysis, percutaneous trans-luminal angioplasty (PTA) and coronarybypass operations; the prevention of re-thrombosis after microsurgeryand vascular surgery in general.

[0214] Further indications include the therapeutic and/or prophylactictreatment of disseminated intravascular coagulation caused by bacteria,multiple trauma, intoxication or any other mechanism; anticoagulanttreatment when blood is in contact with foreign surfaces in the bodysuch as vascular grafts, vascular stents, vascular catheters, mechanicaland biological prosthetic valves or any other medical device; andanticoagulant treatment when blood is in contact with medical devicesoutside the body such as during cardiovascular surgery using aheart-lung machine or in haemodialysis.

[0215] In addition to its effects on the coagulation process, thrombinis known to activate a large number of cells (such as neutrophils,fibroblasts, endothelial cells and smooth muscle cells). Therefore, thecompounds of the invention may also be useful for the therapeutic and/orprophylactic treatment of idiopathic and adult respiratory distresssyndrome, pulmonary fibrosis following treatment with radiation orchemotherapy, septic shock, septicemia, inflammatory responses, whichinclude, but are not limited to, edema, acute or chronic atherosclerosissuch as coronary arterial disease, cerebral arterial disease, peripheralarterial disease, reperfusion damage, and restenosis after percutaneoustrans-luminal angioplasty (PTA).

[0216] Compounds of the invention that inhibit trypsin and/or thrombinmay also be useful in the treatment of pancreatitis.

[0217] According to a further aspect of the present invention, there isprovided a method of treatment of a condition where inhibition ofthrombin is required which method comprises administration of atherapeutically effective amount of a compound of the invention, or apharmaceutically acceptable salt thereof, to a person suffering from, orsusceptible to such a condition.

[0218] The compounds of the invention will normally be administeredorally, intravenously, subcutaneously, buccally, rectally, dermally,nasally, tracheally, bronchially, by any other parenteral route or viainhalation, in the form of pharmaceutical preparations comprising activecompound either as a free base, or a pharmaceutical acceptable non-toxicorganic or inorganic acid addition salt, in a pharmaceuticallyacceptable dosage form. Depending upon the disorder and patient to betreated and the route of administration, the compositions may beadministered at varying doses.

[0219] The compounds of the invention may also be combined and/orco-administered with any antithrombotic agent with a different mechanismof action, such as the antiplatelet agents acetylsalicylic acid,ticlopidine, clopidogrel, thromboxane receptor and/or synthetaseinhibitors, fibrinogen receptor antagonists, prostacyclin mimetics andphosphodiesterase inhibitors and ADP-receptor (P₂T) antagonists.

[0220] The compounds of the invention may further be combined and/orco-administered with thrombolytics such as tissue plasminogen activator(natural, recombinant or modified), streptokinase, urokinase,prourokinase, anisoylated plasminogen-streptokinase activator complex(APSAC), animal salivary gland plasminogen activators, and the like, inthe treatment of thrombotic diseases, in particular myocardialinfarction.

[0221] According to a farther aspect of the invention there is thusprovided a pharmaceutical formulation including a compound of theinvention, in admixture with a pharmaceutically acceptable adjuvant,diluent or carrier.

[0222] Suitable daily doses of the compounds of the invention intherapeutical treatment of humans are about 0.001-100 mg/kg body weightat peroral administration and 0.001-50 mg/kg body weight at parenteraladministration.

[0223] The compounds of the invention have the advantage that they maybe more efficacious, be less toxic, be longer acting, have a broaderrange of activity, be more potent, produce fewer side effects, be moreeasily absorbed than, or that they may have other useful pharmacologicalproperties over, compounds known in the prior art.

Biological Tests

[0224] Test A

[0225] Determination of Thrombin Clotting Time (TT)

[0226] The inhibitor solution (25 μL) was incubated with plasma (25 μL)for three minutes. Human thrombin (T 6769; Sigma Chem Co) in buffersolution, pH 7.4 (25 μL) was then added and the clotting time measuredin an automatic device (KC 10; Amelung).

[0227] The clotting time in seconds was plotted against the inhibitorconcentration, and the IC₅₀TT was determined by interpolation.

[0228] IC₅₀TT is the concentration of inhibitor in the test that doublesthe thrombin clotting time for human plasma.

[0229] Test B

[0230] Determinaton of Thrombin Inhibition with a Chromogenic, RoboticAssay

[0231] The thrombin inhibitor potency was measured with a chromogenicsubstrate method, in a Plato 3300 robotic microplate processor (RosysAG, CH-8634 Hombrechtikon, Switzerland), using 96-well, half volumemicrotitre plates (Costar, Cambridge, Mass., USA; Cat No 3690). Stocksolutions of test substance in DMSO (72 μL), 1 mmol/L, were dilutedserially 1:3 (24+48 μL) with DMSO to obtain ten differentconcentrations, which were analysed as samples in the assay. 2 μL oftest sample was diluted with 124 μL assay buffer, 12 μL of chromogenicsubstrate solution (S-2366, Chromogenix, Mölndal, Sweden) in assaybuffer and finally 12 μL of (α-thrombin solution, (Human α-thrombin,Sigma Chemical Co.) both in assay buffer, were added, and the samplesmixed. The final assay concentrations were: test substance 0.00068-13.3μmol/L, S-2366 0.30 mmol/L, α-thrombin 0.020 NIHU/mL. The linearabsorbance increment during 40 minutes incubation at 37° C. was used forcalculation of percentage inhibition for the test samples, as comparedto blanks without inhibitor. The IC₅₀-robotic value, corresponding tothe inhibitor concentration which caused 50% inhibition of the thrombinactivity, was calculated from a log dose vs. % inhibition curve.

[0232] Test C

[0233] Determinaton of the Inhibition Constant K₁ for Human Thrombin

[0234] K₁ determinations were made using a chromogenic substrate method,performed at 37° C. on a Cobas Bio centrifugal analyser (Roche, Basel,Switzerland). Residual enzyme activity after incubation of humanα-thrombin with various concentrations of test compound was determinedat three different substrate concentrations, and was measured as thechange in optical absorbance at 405 nm.

[0235] Test compound solutions (100 μL; normally in buffer or salinecontaining BSA 10 g/L) were mixed with 200 μL of human α-thrombin (SigmaChemical Co) in assay buffer (0.05 mol/L Tris-HCl pH 7.4, ionic strength0.15 adjusted with NaCl) containing BSA (10 g/L), and analysed assamples in the Cobas Bio. A 60 μL sample, together with 20 μL of water,was added to 320 μL of the substrate S-2238 (Chromogenix AB, Mölndal,Sweden) in assay buffer, and the absorbance change (ΔA/min) wasmonitored. The final concentrations of S-2238 were 16, 24 and 50 μmol/Land of thrombin 0.125 NIH U/ml.

[0236] The steady state reaction rate was used to construct Dixon plots,i.e. diagrams of inhibitor concentration vs. 1/(ΔA/min). For reversible,is competitive inhibitors, the data points for the different substrateconcentrations typically form straight lines which intercept atx=−K_(i).

[0237] Test D

[0238] Determination of Activated Partial Thromboplastin Time (APTT)

[0239] APTT was determined in pooled normal human citrated plasma withthe reagent PTT Automated 5 manufactured by Stago. The inhibitors wereadded to the plasma (10 μL inhibitor solution to 90 μl plasma) andincubated with the APTT reagent for 3 minutes followed by the additionof 100 μL of calcium chloride solution (0.025M) and APTT was determinedin the mixture by use of the coagulation analyser KC10 (Amelung)according to the instructions of the reagent producer. The clotting timein seconds was plotted against the inhibitor concentration in plasma andthe IC₅₀APTT was determined by interpolation.

[0240] IC₅₀APTT is defined as the concentration of inhibitor in humanplasma that doubled the Activated Partial Thromboplastin Time.

[0241] Test E

[0242] Determination of Thrombin Time Ex Vivo

[0243] The inhibition of thrombin after oral or parenteraladministration of the compounds of formula I and Ia, dissolved inethanol:Solutol™:water (5:5:90), were examined in conscious rats which,one or two days prior to the experiment, were equipped with a catheterfor blood sampling from the carotid artery. On the experimental dayblood samples were withdrawn at fixed times after the administration ofthe compound into plastic tubes containing 1 part sodium citratesolution (0.13 mol per L.) and 9 parts of blood. The tubes werecentrifuged to obtain platelet poor plasma. The plasma was used fordetermination of thrombin time as described below.

[0244] The citrated rat plasma, 100 μl, was diluted with a salinesolution, 0.9%, 100 μl, and plasma coagulation was started by theaddition of human thrombin (T 6769, Sigma Chem Co, USA) in a buffersolution, pH 7.4, 100 μl. The clotting time was measured in an automaticdevice (KC 10, Amelumg, Germany).

[0245] Where a compound of formula Ia was administered, concentrationsof the appropriate active thrombin inhibitor of formula I in the ratplasma were estimated by the use of standard curves relating thethrombin time in the pooled citrated rat plasma to known concentrationsof the corresponding “active” thrombin inhibitor dissolved in saline.

[0246] Based on the estimated plasma concentrations of the activethrombin inhibitor of formula I (which assumes that thrombin timeprolongation is caused by the aforementioned compound) in the rat, thearea under the curve after oral and/or parenteral administration of thecorresponding prodrug of formula Ia was calculated (AUCpd) using thetrapezoidal rule and extrapolation of data to infinity.

[0247] The bioavailability of the active thrombin inhibitor of formula Iafter oral or parenteral administration of the prodrug of formula Ia wascalculated as below:

[(AUCpd/dose)/(AUCactive,parenteral/dose]×100

[0248] where AUCactive,parenteral represents the AUC obtained afterparenteral administration of the corresponding active thrombin inhibitorof formula I to conscious rats as described above.

[0249] Test F

[0250] Determination of Thrombin Time in Urine Ex Vivo

[0251] The amount of the active thrombin inhibitor of formula I that wasexcreted in urine after oral or parenteral administration of thecompounds of the invention, dissolved in ethanol:Solutol™:water(5:5:90), was estimated by determination of the thrombin time in urineex vivo (assuming that thrombin time prolongation is caused by theaforementioned compound).

[0252] Conscious rats were placed in metabolism cages, allowing separatecollection of urine and faeces, for 24 hours following oraladministration of compounds of the invention. The thrombin time wasdetermined on the collected urine as described below.

[0253] Pooled normal citrated human plasma (100 μL) was incubated withthe concentrated rat urine, or saline dilutions thereof, for one minute.Plasma coagulation was then initiated by the administration of humanthrombin (T 6769, Sigma Chem Company) in buffer solution (pH 7.4; 100μL). The clotting time was measured in an automatic device (KC 10;Amelung).

[0254] The concentrations of the active thrombin inhibitor of formula Iin the rat urine were estimated by the use of standard curves relatingthe thrombin time in the pooled normal citrated human plasma to knownconcentrations of the aforementioned active thrombin inhibitor dissolvedin concentrated rat urine (or saline dilutions thereof). By multiplyingthe total rat urine production over the 24 hour period with theestimated mean concentration of the aforementioned active inhibitor inthe urine, the amount of the active inhibitor excreted in the urine(AMOUNTpd) could be calculated.

[0255] The bioavailability of the active thrombin inhibitor of formula Iafter oral or parenteral administration of the prodrug, was calculatedas below:

[(AMOUNTpd/dose)/(AMOUNTactive,parenteral/dose]×100

[0256] where AMOUNTactive,parenteral represents the amount excreted inthe urine after parenteral administration of the corresponding activethrombin inhibitor of formula I to conscious rats as described above.

[0257] The invention is illustrated by way of the following examples.The amino acids Pro and Aze are defined as the S-isomers if nototherwise specified. The examples were obtained as diastereoisomers ifnot otherwise specified.

EXAMPLES General Experimental Procedures

[0258] Mass spectra were recorded on a Finnigan MAT TSQ 700 triplequadrupole mass spectrometer equipped with an electrospray interface(FAB-MS) and VG Platform II mass spectrometer equipped with anelectrospray inter-.ace (LC-MS). ¹H NMR and ¹³C NMR measurements wereperformed on BRUKER ACP 300 and Varian UNITY plus 400, 500 and 600spectrometers, operating at ¹H frequencies of 300.13, 399.96, 499.82 and599.94 MHz respectively, and at ¹³C frequencies of 75.46, 100.58, 125.69and 150.88 MHz respectively. Flash chromatography was carried out onsilica gel (230-400 mesh). Preparative RPLC was performed on reversephase columns (250 mm, 20 or 50 mm; 5 to 7 μM phase Chromasil C8) withflow rates of 10 to 50 mL/min using a UV detector (270 to 280 nm).

Example 1 (S)- or (R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Pro-Pab

[0259] (i) 1-Hydroxy-7-methoxytetralin-1-yl-carboxylic Acid, MethylEster

[0260] The sub-title compound was prepared according to the methoddescribed by C. F. Bigge et al (J. Med. Chem (1993) 36, 1977) using7-methoxytetralone (1.0 g; 5.67 mmol) and methanol instead of ethanol.Yield: 1.22 g (90%).

[0261]¹H-NMR (300 MHz; CDCl₃ ): δ 7.05 (d, 1H), 6.80 (d, 1H), 6.65 (s,1H), 3.80 (s, 3H), 3.75 (s, 3H), 2.85-2.65 (m, 2H), 2.25-1.90 (m, 4H)

[0262] (ii) 1-Hydroxy-7-methoxytetralin-1-yl-1-carboxylic Acid

[0263] 1-Hydroxy-7-methoxytetralin-1-yl-carboxylic acid, methyl ester(1.16 g; 4.9 mmol; from step (i) above) was dissolved in THF (10 mL) andlithium hydroxide (0.41 g; 9.8 mmol) was added to the resultantsolution, followed by water (4 mL). The reaction mixture was stirred atroom temperature for 3 h, the THF was evaporated, and the aqueous phasewas washed with methylene chloride. The reaction mixture was acidifiedwith HCl (2M) and then saturated with NaCl(s). After extraction withCH₂Cl₂, the organic phase was dried and concentrated. Yield: 765 mg(70%).

[0264] LC-MS (m/z) 221 (M−1)⁻ ¹H-NMR (400 MHz; CDCl₃): δ 7.07 (d, 1H),6.82 (dd, 1H), 6.77 (d, 1H), 3.76 (s, 3H), 2.83-2.71 (m, 2H), 2.32-2.21(m, 1H), 2.12-1.88 (m, 3H)

[0265] (iii) (R)- and(S)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Pro-Pab(Z)

[0266] A solution of 1-hydroxy-7-methoxytetralin-1-yl-carboxylic acid(222 mg; 1.0 mmol; from step (ii) above), H-Pro-Pab(Z) (499 mg; 1.1mmol; prepared according to the method described in International PatentApplication WO 97/02284) and TBTU (353 mg; 1.1 mmol) in DMF (10 ml) wascooled to 0° C., and DIPEA (517 mg, 4.0 mmol) was added. The reactionmixture was stirred at room temperature for 4 days and then the sameamounts of H-Pro-Pab(Z), TBTU and DIPEA were added at 0° C. After 3 daysthe reaction mixture was concentrated and dissolved in water:EtOAc(1:1). The aqueous phase was extracted with EtOAc and the combinedorganic phase was dried (Na₂SO₄) and concentrated. The product waspurified using flash chromatography (EtOAc:EtOH; 100:0 to 95:5). Furtherpurification using preparative RPLC (CH₃CN:0.1M ammonium acetate; 40:60)separated the diastereomers: Compound 1A (faster moving diastereomer; 10mg; 1.7%) and Compound 1B (slower moving diastereomer; 10 mg; 1.7%).Yield: 20 mg (3.4%).

[0267] Compound 1A:

[0268]¹H-NMR (400 MHz; CDCl₃): δ 7.82 (d, 2H), 7.44 (d, 2H), 7.38-7.29(m, 4H), 7.05 (d, 2H), 6.80 (dd, 1H), 6.54 (d, 1H), 5.21 (s, 2H)4.68-4.63 (dd, 1H), 4.45 (m, 2H), 3.71 (s, 3H), 3.12 (m, 1H), 2.83 (m,1H), 2.68-2.53 (m, 2H), 2.22-2.13 (m, 2H), 2.05-1.84 (m, 7H), 1.59-1.50(m, 1H)

[0269] Compound 1B:

[0270]¹H-NMR (400 MHz; CDCl₃): δ 7.82 (d, 2H), 7.43 (d, 2H), 7.37-7.28(m, 4H), 7.02 (d, 2H), 6.77 (dd, 1H), 6.57 (d, 1H), 5.20 (s, 2H)4.58-4.51 (m, 2H), 4.42 (m, 1H), 3.62 (s, 3H), 3.12-3.04 (m, 1H), 2.83(bd, 1H), 2.68-2.58 (m, 1H), 2.55-2.47 (m, 1H), 2.13-1.79 (m, 7H),1.76-1.65 (m, 1H)

[0271] (iv) (S)- or (R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Pro-Pab

[0272] Pd/C (5%; 10 mg) was added to a solution of (R)- or(S)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Pro-Pab(Z) (10 mg; 0.017 mmol;Compound 1A from step (iii) above) in EtOH (5 mL) and HOAc (1 μL, 0.017mmol), and the mixture was hydrogenated for 3 hours at room temperatureand atmospheric pressure. The resultant mixture was filtered throughCelite, the solution was concentrated, water was added and the solutionwas freeze dried, yielding 10 mg (98%; purity 92.2%) of the titlecompound as a white powder.

[0273] LC-MS (m/z) 451 (M+1)⁺ ¹H-NMR (400 MHz; CD₃OD): δ 7.75 (d, 2H),7.57 (d, 2H), 7.08 (d, 1H), 6.83 (dd, 1H), 6.60 (d, 1H), 4.63-4.40 (m,3H), 3.69 (s, 3H), 3.43-3.35 (m, 1H), 2.88-2.67 (m, 3H), 2.23-2.11 (m,2H), 2.20-1.77 (m, 8H), 1.63-1.51 (m, 1H)

Example 2 (R)- or (S)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Pro-Pab

[0274] The title compound was prepared according to the method describedin Example 1(iv) above from (R)- or(S)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Pro-Pab(Z) (10 mg; 0.017 mmol;Compound 1B from Example 1(iii) above). Yield: 10 mg (98%; purity80.4%).

[0275] LC-MS (m/z) 451 (M+1)⁺ ¹H-NMR (400 MHz; CD₃OD) δ 7.78 (d, 2H),7.63 (d, 2H), 7.04 (d, 1H), 6.78 (dd, 1H), 6.75 (d, 1H), 4.67-4.48 (m,3H), 3.68 (s, 3H), 3.30-3.23 (m, 1H), 2.86-2.61 (m, 3H), 2.23-1.71 (m,11H)

Example 3 (S)- or (R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab×HOAc

[0276] (i) (S)- and (R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Z)

[0277] TBTU (0.584 g; 1.7 mmol), followed by DIPEA (0.200 g; 1.55 mmol)were added to an ice-cold solution of1-hydroxy-7-methoxytetralin-1-yl-carboxylic acid (0.345 g; 1.55 mmol;see Example 1(ii) above) in DMF (10 mL). After stirring at 0° C. for 15minutes, H-Aze-Pab(Z)×2HCl (0.750 g; 1.7 mmol; prepared according to themethod described in International Patent Application WO 97/02284) andDIPEA (0.603 g; 4.65 mmol) were added and the mixture was stirred at RTfor 4 days. The DMF was evaporated, and the resultant material waspartitioned between water and EtOAc. The organic layer was separated,the water phase was extracted 3 times with EtOAc, and the combinedorganic layer was dried (Na₂SO₄) and concentrated. The product, a whitepowder, was further purified using preparative RPLC (CH₃CN:0.1M ammoniumacetate; 46:54) yielding 122 mg (28%) of a faster moving fraction(Compound 3A) and 63 mg (14%) of a slower moving fraction (Compound 3B).

[0278] Compound 3A:

[0279] LC-MS (m/z) 571 (M+1)⁺ ¹H-NMR (400 MHz; CDCl₃): (complex due torotamers) δ 8.22 (t, 0.5H, rotamer); 7.94 (t, 0.5H, rotamer); 7.83 (t,1H); 7.45-7.3 (m, 9H); 7.4 (t, 1H); 6.80 (m, 1H); 4.93 (m, 1H); 4.55 (m,5H); 3.76 (s, 3H); 3.0 (m, 2H); 2.8 (m, 2H); 2.6 (m, 2H); 2.5 (m, 1H);2.38 (m, 1H); 2.25 (m, 1H); 2.0-1.8 (m, 9H)

[0280] (ii) (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab×HOAc

[0281] Prepared according to the method described in Example 1(iv) from(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Z) (0.058 g;0.1 mmol; Compound 3A from step (i) above), HOAc (5.8 μL; 0.1 mmol), andPd/C (5%; 50 mg) in EtOH (5 ml). Yield 15 mg (59%).

[0282] LC-MS (m/z) 437 (M+1)⁺ ¹H-NMR (400 MHz; D₂O): δ 7.65 (d, 2H);7.47 (d, 2H); 7.16 (d, 1H); 6.90 (d, 1H); 6.71 (d, 1H); 4.91 (dd, 1H);4.40 (m, 1H); 4.15 (m, 1H); 3.94 (m, 1H); 3.60 (s, 3H); 2.75 (m, 3H);2.53 (m, 1H); 2.1 (m, 2H); 2.0-1.75 (m, 7H) ¹³C-NMR (100 MHz; CDCl₃) δ182.5; 178.3; 174.0

Example 4 (R)- or (S)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab

[0283] (i) 4-(Amino, Methoxyiminometyl)benzyl Amine (H-Pab(OMe))

[0284] Platinum oxide (200 mg) was added to a solution of 4-(amino,methoxyiminomethyl)benzyl azide (10 g; 0.049 mol; prepared according tothe method described in WO 94/29336) in 200 mL of ethanol. The mixturewas hydrogenated at atmospheric pressure for 8 h, filtered through Hyfloand concentrated. The crude product was used directly in the followingstep.

[0285]¹H-NMR (400 MHz; CD₃OD): δ 7.60 (d, 2H); 7.37 (d, 2H); 3.81 (s,3H); 3.80 (s, 2H).

[0286] (ii) Boc-Aze-Pab(OMe)

[0287] DIPEA (17.5 mL; 105 mmol) was added to an ice-cold solution ofBoc-Aze-OH (9.7 g; 48 mmol) and H-Pab(OMe) (9.4 g; 52 mmol; from step(i) above) and TBTU (18.5 g; 58 mmol) in DMF (100 mL), and the mixturewas stirred overnight at RT. The resultant mixture was poured onto water(50 mL), the pH was adjusted to ca 9, and the mixture was extractedthree times with EtOAc. The combined organic layer was washed withNaHCO₃/aq, water and brine, dried (Na₂SO₄), and concentrated. The crudeproduct was purified using flash chromatography (Si-gel, EtOAc). Yield:11.9 g (69%).

[0288]¹H-NMR (400 MHz; CDCl₃): δ 7.60 (d, 2H); 7.31 (d, 2H); 4.78 (b,2H); 4.69 (m, 1H); 4.50 (b, 2H); 3.92 (s+m, 4H); 3.79 (m, 1H); 2.46 (b,2H); 1.42 (s, 9H)

[0289] (iii) H-Aze-Pab(OMe)×2HCl

[0290] A solution of Boc-Aze-Pab(OMe) (9.4 g; 26 mmol; from step (ii)above) in EtOAc (250 mL) was saturated with HCl(g). EtOH (abs; 125 mL)was added to the resultant emulsion and the mixture was sonicated for 10min. EtOAc was added until the solution became turbid, whereafter thesub-title product soon crystallized. Yield: 6.7 g (77%).

[0291] LC-MS (m/z) 263 (M+1)⁺ ¹H-NMR (400 MHz; CD₃OD): δ 7.74 (d, 2H);7.58 (d, 2H); 5.13 (t, 1H); 4.57 (m, 2H); 4.15 (m, 2H); 3.97 (s+m, 4H);2.87 (m, 1H); 2.57 (m, 1H) ¹³C-NMR (75 MHz; CDCl₃): (carbonyl and/oramidine carbons) δ 168.9; 168.8; 161.9

[0292] (iv) 1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(OMe)

[0293] H-Aze-Pab(OMe) (0.587 g; 1.85 mmol; from step (iii) above), TBTU(0.594 g; 1.85 mmol) and DIPEA (0.87 g; 6.73 mmol) were added, in thatorder, to an ice-cold solution of1-hydroxy-7-methoxytetralin-1-yl-carboxylic acid (0.374 g; 1.68 mmol;see Example 1(ii) above) in CH₃CN. The resultant mixture was stirred atRT for 6 days. The solution was concentrated and the crude material waspurified using preparative HPLC (CH₃CN:0.1M ammonium acetate; 25:75),which procedure separated diastereomers, yielding 122 mg (31.2%) of afaster moving diastereomer Compound 4A) and 120 mg (30.7%) of a slowermoving diastereomer Compound 4B).

[0294] Compound 4B:

[0295] LC-MS (m/z) 466 (M+1)⁺ ¹H-NMR (500 MHz; CDCl₃): δ 8.08 (t, 1H);7.63 (d, 2H); 7.35 (d, 2H); 7.02 (d, 1H); 6.80 (dd, 1H); 6.57 (d, 1H);4.90 (dd, 1H); 4.79 (b, 2H); 4.53 (m, 2H); 3.91 (s, 3H); 3.65 (s+m, 4H);2.97 (q, 1H); 2.81 (bd, 1H); 2.59 (m, 1H); 2.49 (m, 1H); 2.38 (m, 1H);2.03-1.85 (m, 4H)

[0296] (v) (R)- or (S)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab

[0297] The title compound was prepared according to the method describedin Example 1(iv) above from1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(OMe) (20 mg; 0.043 mmol;Compound 4B from step (iv) above), AcOH (3 mg; 0.05 mmol) and Pd/C (10%;20 mg) in EtOH (5 mL). Yield 19 mg (89%).

[0298] LC-MS (m/z) 436 (M+1)⁺ ¹H-NMR (400 MHz; D₂O): δ 7.79 (d, 2H);7.55 (m, 2H); 7.20 (d, 1H); 6.95 (m, 1H); 6.79 (d, 1H); 4.92 (dd, 1H);4.58 (m, 2H); 4.18 (m, 2H); 3.77 (s, 3H); 3.63 (m, 2H); 2.8 (m, 3H); 2.6(m, 2H); 2.1 (m, 4H); 1.9 (m, 2H) ¹³C-NMR (75 MHz; CDCl₃): (carbonyland/or amidine carbons) δ 177.9; 173.8; 167.6

Example 5 1-Hydroxy-5-methoxytetralin-1-yl-C(O)-Aze-Pab×HOAc

[0299] (i) 1-Hydroxy-5-methoxytetralin-1-yl-carboxylic Acid, MethylEster

[0300] The sub-title compound was prepared according to a methoddescribed by Bigge et al (J. Med. Chem. (1993) 36, 1977) from5-methoxytetralone (1.0 g; 5.67 mmol), Me₃SiCN (0.619 g; 6.24 mmol), andZnI₂ (8 mg; cat.). Yield 1.11 g (83%).

[0301]¹H-NMR (500 MHz; CDCl₃): δ 7.16 (m, 1H); 6.91 (d, 1H); 6.76 (t,1H); 6.45 (br, 1H); 5.97 (br, 1H); 3.815 (s, 2H); 3.81 (s, 3H); 2.88 (m,1H); 2.56 (m, 2H); 2.14 (m, 2H); 1.95 (m, 2H)

[0302] (ii) 1-Hydroxy-5-methoxytetralin-1-yl-carboxylic Acid

[0303] Prepared according to the method described in Example 1(ii) abovefrom 1-hydroxy-5-methoxytetralin-1-yl-carboxylic acid, methyl ester(1.11 g; 4.7 mmol; from step (i) above) and LiOH.H₂O (0.395 g; 9.4mmol). Yield 460 mg (36%).

[0304]¹H-NMR (400 MHz; CDCl₃): δ 7.18 (m, 1H); 6.86 (d, 1H); 6.79 (d,1H); 3.82 (s, 3H); 2.86 (dt, 1H); 2.58 (m, 1H); 2.20 (m, 1H); 2.10-1.85(m, 4H)

[0305] (iii) 1-Hydroxy-5-methoxytetralin-1-yl-C(O)-Aze-Pab(Z)

[0306] TBTU (0.528 g; 1.64 mmol) was added to an ice-cold solution of1-hydroxy-5-methoxytetralin-1-yl-carboxylic acid (0.332 g; 1.49 mmol;from step (ii) above) in CH₃CN (15 mL). The mixture was stirred at 0° C.for 2 h, and H-Aze-Pab(Z)×2HCl (0.656 g; 1.49 mmol) and DIPEA (0.599 g;3.1 mmol) were added. The resultant mixture was stirred at RT overnight,and the solution was concentrated. The crude product was purified usingpreparative RPLC (CH₃CN:0.1M ammonium acetate; 40:60). Yield 140 mg(16%).

[0307] LC-MS (m/z) 571 (M+1)⁺ ¹H-NMR (400 MHz; CDCl₃ ): δ 7.82 (dd, 2H);7.43 (d, 2H); 7.33 (t, 2H); 7.27 (m, 3H); 6.73 (m, 2H); 5.20 (s, 2H);4.89 (m, 1H); 4.60-4.40 (m, 2H); 3.80 (s, 3H); 3.62 (m, 1H); 2.94 (m,2H); 2.34 (m, 2H); 1.95-1.8 (m, 4H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyland/or amidine carbons) δ 184.2; 179.0; 178.6; 172.3; 171.6; 168.9

[0308] (iv) 1-Hydroxy-5-methoxytetralin-1-yl-C(O)-Aze-Pab×HOAc

[0309] Prepared according to the method described in Example 1(iv) from1-hydroxy-5-methoxytetralin-1-yl-C(O)-Aze-Pab(Z) (47 mg; 0.082 mmol;from step (iii) above), AcOH (5 mg; 0.082 mmol) and Pd/C (5%; 20 mg) inEtOH (5 mL). Yield 37 mg (100%).

[0310] LC-MS (m/z) 437 (M+1)⁺ ¹H-NMR (400 MHz; D₂O): δ 7.76 (dd, 2H);7.54 (dd, 2H); 7.27 (m, 1H); 7.01 (t, 1H); 6.90 (dd, 1H); 4.91 (dd, 1H);4.5 (m, 1H); 4.20 (m, 1H); 3.87 (s, 3H); 3.63 (m, 1H); 2.90 (m, 1H);2.7-2.4 (m, 2H); 2.24 (m, 1H) ¹³C-NMR (100 MHz; CDCl₃ ): (carbonyland/or amidine carbons) δ 181.5; 177.5; 177.2; 173.1; 173.0; 166.7

Example 6 1-Hydroxy-5,7-dimethyltetralin-1-yl-C(O)-Aze-Pab×HOAc

[0311] (i) 1-Hydroxy-5,7-dimethyltetralin-1-yl-carboxylic Acid, MethylEster

[0312] The sub-title compound was prepared according to a methoddescribed by Bigge et al (J. Med. Chem. (1993) 36, 1977) from5,7-dimethyltetralone (1.0 g; 5.74 mmol), Me₃SiCN (0.626 g; 6.31 mg),and ZnI₂ (8 mg; cat.). Yield 1.24 g (92%).

[0313]¹H-NMR (400 MHz; CDCl₃): δ 6.94 (s, 1H); 6.81 (s, 1H); 3.77 (s,3H); 2.82 (t, 1H); 2.73 (m, 1H); 2.60 (m, 3H); 2.25 (s, 3H); 2.21 (s,3H); 2.00 (m, 3H)

[0314] (ii) 1-Hydroxy-5,7-dimethyltetralin-1-yl-carboxylic Acid

[0315] Prepared according to the method described in Example 1(ii) from1-hydroxy-5,7-dimethyltetralin-1-yl-carboxylic acid, methyl ester (1.24g; 5.27 mmol; from step (i) above) and LiOH.H₂O (0.443 mg; 10.6 mmol).Yield 0.629 g (50%).

[0316] LC-MS (m/z) 437 (M−1)⁻ ¹H-NMR (400 MHz; CDCl₃): δ 6.97 (s, 1H);6.92 (s, 1H); 2.72 (m, 1H); 2.60 (m, 1H); 2.27 (s, 3H); 2.22 (s, 3H);2.06 (m, 2H); 1.95 (m, 2H)

[0317] (iii) 1-Hydroxy-5,7-dimethyltetralin-1-yl-C(O)-Aze-Pab(OMe)

[0318] Prepared according to the method described in Example 4(iv) abovefrom 1-hydroxy-5,7-dimethyltetralin-1-yl-carboxylic acid (0.20 g; 0.91mmol; from step (ii) above), H-Aze-Pab(OMe)×1.5HCl (0.317 g; 1.0 mmol;see Example 4(iii) above), TBTU (0.321 g; 1.0 mmol) and DIPEA (0.469 g;3.63 mmol). The solution was concentrated and the remainder was purifiedusing preparative RPLC (CH₃CN:0.1M ammonium acetate; 30:70). Thefractions of interest were concentrated and were then extracted (×3)with EtOAc. The combined organic layer was dried (Na₂SO₄) andconcentrated. The dry product was dissolved in a small amount ofwater/CH₃CN and freeze dried. Yield 40 mg (9.5%).

[0319] LC-MS (m/z) 463 (M+1)⁺ ¹H-NMR (400 MHz; CDCl₃): (complex due todiastereomers/rotamers) δ 8.19 (bt, 0.5H, rotamer); 7.91 (bt, 0.5H,rotamer); 7.61 (dd, 2H); 7.35 (d, 1H); 7.28 (d, 1H); 6.93 (s, 0.5H,rotamer); 6.91 (s, 0.5H, rotamer); 6.80 (s, 0.5H, rotamer); 6.70 (s,0.5H, rotamer); 4.91 (m, 2H); 4.79 (b, 2H); 4.50 (m, 3H); 3.91 (d, 3H);3.74 (m, 0.5H, rotamer); 3.61 (m, 0.5H, rotamer); 2.78 (bd, 1H); 2.57(m, 1H); 2.38 (m, 2H); 2.26 (m, 2H); 2.19 (d, 6H); 1.95 (m, 3H) ¹³C-NMR(100 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ 179.2; 178.9;171.6; 171.4

[0320] (iv) 1-Hydroxy-5,7-dimethyltetralin-1-yl-C(O)-Aze-Pab×HOAc

[0321] Prepared according to the method described in Example 1(iv) abovefrom 1-hydroxy-5,7-dimethyltetralin-1-yl-C(O)-Aze-Pab(OMe) (20 mg; 0.043mmol; from step (iii) above), HOAc (2.6 mg; 0.043 mmol) and Pd/C (10%;10 mg). Yield 20 mg (94%).

[0322] LC-MS (m/z) 435 (M+1)⁺ ¹H-NMR (400 MHz; D₂O): δ 7.79 (dd, 1H);7.70 (d, 1H); 7.53 (m, 2H); 7.07 (d, 1H); 6.92 (s, 1H); 4.91 (m, 1H);4.17 (m, 1H); 3.76 (m, 0.5H, rotamer); 3.60 (m, 0.5H, rotamer); 2.80 (d,1H); 2.55 (m, 2H); 2.23 (s, 3H); 2.07 (m, 2H); 1.95 (s, 6H) ¹³C-NMR (100MHz; D₂O): (carbonyl and/or amidine carbons) δ 177.9; 173.3

Example 7 1-Hydroxy-7-aminotetralin-1-yl-C(O)-Aze-Pab×HOAc

[0323] (i) 1-Hydroxy-7-nitrotetralin-1-yl-carboxylic Acid, Methyl Ester

[0324] The sub-title compound was prepared according to a methoddescribed by Bigge et al (J. Med. Chem. (1993) 36, 1977) from7-nitrotetralone (2.0 g; 10.5 mmol), Me₃SiCN (1.14 g; 11.5 mg) and ZnI₂(8 mg; cat.). Yield 2.87 g (100%) (over 3 steps).

[0325]¹H-NMR (400 MHz; CDCl₃): δ 8.16 (dd, 1H); 8.04 (m, 1H); 7.36 (dd,1H); 3.73 (s, 3H); 2.92 (m, 2H); 2.30 (m, 1H); 2.00 (m, 3H)

[0326] (ii) 1-Hydroxy-7-nitrotetralin-1-yl-carboxylic Acid

[0327] The sub-title compound was prepared according to the methoddescribed in Example 1(ii) above from1-hydroxy-7-nitrotetralin-1-yl-carboxylic acid, methyl ester (2.0 g; 8.3mmol; from step (i) above) and LiOH.H₂O (0.7 g; 16.6 mmol). Yield 1.72 g(88%).

[0328] LC-MS (m/z) 236 (M+1)⁺ ¹H-NMR (400 MHz; CDCl₃): δ 8.10 (dd, 1H);8.05 (m, 1H); 7.30 (d, 1H); 2.92 (m, 2H); 2.30 (m, 1H); 2.15-1.85 (m,3H)

[0329] (iii) 1-Hydroxy-7-nitrotetralin-1-yl-C(O)-Aze-Pab(Z)

[0330] HATU (0.352 g; 0.93 mmol) and DIPEA (0.200 g; 1.55 mmol) wereadded to an ice-cold solution of1-hydroxy-7-nitrotetralin-1-yl-carboxylic acid (0.200 g; 0.84 mmol; fromstep (ii) above) in DMF (5 mL). After stirring at 0° C. for 15 minutes asolution of H-Aze-Pab(Z)×2HCl (0.408 g; 0.93 -mmol) and2,4,6-trimethylpyridine (0.409 g; 3.37 mmol) in 5 mL of DMF was added at0° C., and the mixture was stirred at RT overnight. The DMF wasevaporated, and the crude product was purified using preparative RPLC(CH₃CN:0.1 M ammonium acetate; 40:60). The product was further purifiedusing HPLC (CH₃CN:0.1M ammonium acetate; 46:54), yielding 214 mg (44%)of the sub-title compound.

[0331] LC-MS (m/z) 586 (M+1)⁺ ¹H-NMR (400 MHz; CDCl₃): δ 8.1 (m, 2H);7.77 (d, 1H); 7.71 (d, 1H); 7.40 (d, 2H); 7.32 (t, 2H); 7.27 (m, 2H);7.18 (d, 1H); 4.88 (m, 1H); 4.54 (m, 0.5H, rotamer); 4.45 (dd, 0.5H,rotamer); 4.34 (m, 1H); 3.94 (m, 0.5H, rotamer); 3.82 (m, 0.5H,rotamer); 3.17 (m, 1H); 2.90 (t, 1H); 2.73 (m, 1H); 2.45-2.20 (m, 2H);2.05-1.85 (m, 5H)

[0332] (iv) 1-Hydroxy-7-aminotetralin-1-yl-C(O)-Aze-Pab×HOAc

[0333] The title compound was prepared according to the method describedin Example 1(iv) above from1-hydroxy-7-nitrotetralin-1-yl-C(O)-Aze-Pab(Z) (0.064 g; 0.11 mmol; fromstep (iii) above), HOAc (6.3 μL; 0.11 mmol), and Pd/C (32 mg). The solidcrude product was dissolved in water, and the water solution was freezedried to yield 40 mg (76%).

[0334] LC-MS (m/z) 422 (M+1)⁺ ¹H-NMR (400 MHz; D₂O): δ 7.75 (m, 2H);7.53 (dd, 2H); 7.07 (d, 1H); 6.82 (bt, 1H); 6.67 (b, 1H); 4.93 (m, 1H);4.6-4.4 (m, 2H); 4.29 (m, 0.5H, rotamer); 4.18 (m, 1H); 3.7 (m, 1H);2.8-2.5 (, 3H) ¹³C-NMR (100 MHz; CDCl₃) (carbonyl and/or amidinecarbons) δ 178.4; 178.1; 173.9; 173.8; 167.5

Example 8 1-Hydroxytetralin-1-yl-C(O)-Aze-Pab×HOAc

[0335] (i) 1-Hydroxytetralin-1-yl-carboxylic Acid, Methyl Ester

[0336] The sub-title compound was prepared according to a methoddescribed by Bigge et al (J. Med. Chem. (1993) 36, 1977) from tetralone(2.0 g; 13.7 mmol), Me₃SiCN (1.49 g; 15 mmol) and ZnI₂ (8 mg; cat.).Yield 2.5 g (88%).

[0337] (ii) 1-Hydroxytetralin-1-yl-carboxylic Acid

[0338] The sub-title compound was prepared according to the methoddescribed in Example 1(ii) above from 1-hydroxytetralin-1-yl-carboxylicacid, methyl ester (2.5 g; 12.1 mmol; from step (i) above) and LiOH.H₂O(1.02 g; 24.2 mmol). Yield 400 mg (17%).

[0339]¹H-NMR (400 MHz; CDCl₃): δ 7.18 (m, 4H); 2.92 (t, 0.5H, rotamer);2.78 m, 2H): 2.61 (t, 0.5H, rotamer); 2.22 (m, 1H); 2.1-1.8 (m, 4H)

[0340] (iii) 1-Hydroxytetralin-1-yl-C(O)-Aze-Pab(Z)

[0341] The sub-title compound was prepared according to the methoddescribed in Example 5(iii) above from 1-hydroxytetralin-1-yl-carboxylicacid (0.284 g; 1.50 mmol; from step (ii) above), TBTU (0.531 g; 1.65mmol), H-Aze-Pab(Z)×2HCl (0.660 g; 1.50 mmol) and DIPEA (0.602 g; 3.1mmol). The crude product was purified using preparative RPLC (CH₃CN:0.1Mammonium acetate; 40:60). Yield 70 mg (8.6%).

[0342] LC-MS (m/z) 542 (M+1)⁺ ¹H-NMR (400 MHz; D₂O): (complex due todiastereomers/rotamers) δ 8.15 (t, 0.5H, rotamer); 7.97 (t, 0.5H,rotamer); 7.81 (dd, 2H); 7.43 (dd, 2H); 7.30 (m, 5H); 7.19 (m, 2H); 7.12(m, 2H); 4.88 (m, 1H); 4.47 (m, 2H); 3.79 (m, 0.5H, rotamer); 2.89 (m,2H); 2.66 (m, 1H); 2.50 (m, 0.5H, rotamer); 2.35 (m, 1H); 2.19 (m, 0.5H,rotamer); 1.95 (m, 5H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/oramidine carbons, complex due to diastereomers/rotamers) δ 177.7; 177.4;171.1; 170.5; 170.3; 167.7; 164.4

[0343] (iv) 1-Hydroxytetralin-1-yl-C(O)-Aze-Pab×HOAc

[0344] The title compound was prepared according to the method describedin Example 1(iv) above from 1-hydroxytetralin-1-yl-C(O)-Aze-Pab(Z) (70mg; 0.13 mmol; from step (iii) above), AcOH (5 mg; 0.13 mmol) and Pd/C(5%; 35 mg) in EtOH (5 mL). Yield 61 mg (100%).

[0345] LC-MS (m/z) 407 (M+1)⁺ ¹H-NMR (400 MHz; CD₃OD): δ 7.74 (dd, 2H);7.55 (dd, 2H); 7.29 (d, 1H); 7.15 m, 3H); 4.59 (m, 1H); 4.46 (m, 1H);4.25 (m, 1H); 4.08 (m, 1H); 3.69 (m, 1H); 2.80 (m, 2H); 2.46 (m, 1H);2.3-2.15 (m, 2H) ¹³C-NMR (100 MHz; CD₃OD): (carbonyl and/or amidinecarbons) δ 180.1; 178.0; 177.8; 173.2; 168.2

Example 9 7-Methoxytetralin-1-yl-C(O)-Aze-Pab×HOAc

[0346] (i) 7-Methoxy-3,4-dihydronaphthalen-1-yl-carboxylic Acid, MethylEster

[0347] A solution of 1-hydroxy-7-methoxytetralin-1-yl-carboxylic acid,methyl ester (0.5 g; 2.1 mmol; see Example 1(i) above) in toluene (5 mL)was added to a refluxing solution of p-TsOH (0.6 g; 3.2 mmol) in toluene(10 mL), and the resultant mixture was refluxed for 45 minutes. Aftercooling, the reaction mixture was diluted with ether, washed with waterand NaHCO₃/aq, dried with Na₂SO₄ and concentrated. Yield 392 mg (85%).

[0348]¹H-NMR (500 MHz; CDCl₃): δ 7.46 (d, 1H); 7.19 (t, 1H); 7.06 (d,1H); 6.75 (dd, 1H); 3.84 (s, 3H); 3.83 (s, 3H); 2.69 (t, 2H); 2.38 (m,2H)

[0349] (ii) 7-Methoxy-3,4-dihydronaphthalen-1-yl-carboxylic Acid

[0350] The sub-title compound was prepared according to the methoddescribed in Example 1(ii) above from7-methoxy-3,4-dihydronaphthalen-1-yl-carboxylic acid, methyl ester (0.39g; 1.79 mmol; from step (i) above) and LiOH.H₂O (0.15 g; 3.57 mmol).Yield 148 mg (40%).

[0351] LC-MS (m/z) 203 (M+1)⁺ ¹H-NMR (500 MHz; CDCl₃): δ 7.56 (d, 1H);7.44 (t, 1H); 7.09 (d, 1H); 6.77 (dd, 1H); 3.82 (s, 3H); 2.72 (t, 2H);2.44 (m, 2H)

[0352] (iii) 7-Methoxy-3,4-dihydronaphthalen-1-yl-C(O)-Aze-Pab(Z)

[0353] The sub-title compound was prepared according to the methoddescribed in Example 3(i) above from7-methoxy-3,4-dihydronaphthalen-1-yl-carboxylic acid (0.145 g; 0.71mmol; from step (ii) above), H-Aze-Pab(Z)×2HCl (0.343 g; 0.78 mmol),TBTU (0.251 g; 0.78 mmol) and DIPEA (0.364 g, 2.84 mmol). The crudeproduct was purified using preparative RPLC (CH₃CN:0.1M ammoniumacetate; 54:46). The resultant solution was s concentrated and theaqueous layer was extracted with EtOAc three times. The combined organiclayer was dried (Na₂SO₄) and concentrated, yielding 121 mg (31%).

[0354] LC-MS (m/z) 553 (M+1)⁺ ¹H-NMR (500 MHz; CDCl₃): δ 8.25 (t, 1H);7.82 (d, 2H); 7.43 (d, 2H); 7.37-7.27 (m, 5H); 7.06 (d, 1H); 6.81 (d,1H); 6.72 (dd, 1H); 6.35 (t, 1H); 5.20 (s, 2H); 5.03 (dd, 1H); 4.52 (m,2H); 3.93 (m, 2H); 2.71 (t, 2H); 2.35 (m, 2H)

[0355] (iv) 7-Methoxytetralin-1-yl-C(O)-Aze-Pab×HOAc

[0356] The title compound was prepared according to the method describedin Example 1(iv) above from7-methoxy-3,4-dihydronaphthalen-1-yl-C(O)-Aze-Pab(Z) (88 mg; 0.16 mmol),AcOH (9 mg; 0.16 mmol) and Pd/C (10%; 44 mg). Yield 56 mg (73%), 60:40diastereomeric mixture.

[0357] LC-MS (m/z) 421 (M+1)⁺ ¹H-NMR (500 MHz; D₂O): δ 7.78 (t, 1H);7.65 (d, 1H); 7.55 (dd, 1H); 7.49 (d, 1H); 7.42 (d, 2H); 7.36 (d, 2H);7.16 (m, 1H); 7.05 (d, 1H); 6.84 (dd, 1H); 6.73 (dd, 1H); 6.06 (d, 1H);5.18 (dd, 1H); 4.96 (m, 1H); 4.12 (m, 2H); 3.92 (m, 2H); 3.82 (d, 1H);3.62 (m, 3H); 2.7 (m, 6H); 2.4 (m, 2H); 2.05 (m, 2H); 1.9 (m, 1H); 1.2(m, 2H); 1.5 (m, 1H) ¹³C-NMR (100 MHz; D₂O): (carbonyl and/or amidinecarbons) a 182.3; 179.3; 179.1; 178.8; 173.7; 173.4; 173.0; 166.3

Example 10 (R)- and (S)-7-Methoxy-1-methyltetralin-1-yl-C(O)-Aze-Pab

[0358] (i) 7-Methoxytetralin-1-yl-carboxylic Acid, Methyl Ester

[0359] The sub-title compound was prepared according to the methoddescribed in Example 1(iv) above from7-methoxy-3,4-dihydronaphthalen-1-yl-carboxylic acid, methyl ester (3.3g; 15 mmol; see Example 9(i) above) and Pd/C (10%; 0.5 g). The resultantmixture was filtered through Hyflo and concentrated. The crude productwas purified using flash chromatography (Si-gel; heptane:EtOAc; 4:1).Yield 2.4 g (72%).

[0360]¹H-NMR (400 MHz; CDCl₃): δ 7.04 (d, 1H); 6.77 (dd, 1H); 6.72 (d,1H); 3.82 (t, 1H); 3.78 (s, 3H); 3.73 (s, 3H); 2.75 (m, 3H); 2.14 (m,1H); 1.99 (m, 2H); 1.77 (m, 1H)

[0361] (ii) 7-Methoxy-1-methyltetralin-1-yl-carboxylic Acid Methyl Ester

[0362] 7-Methoxytetralin-1-yl-carboxylic acid, methyl ester (0.4 g; 1.8mmol; from step (i) above) and MeI (0.13 mL, 2.0 mmol) were added to aslurry of NaH (55% in oil; 87 mg; 2.0 mmol) in DMF (5 mL) and themixture was stirred at RT overnight. The resultant mixture was pouredonto water, and the water mixture was extracted with EtOAc:toluene 3times. The combined organic layer was washed with water, dried (Na₂SO₄),and concentrated. Flash chromatography (Si-gel; heptane:EtOAc; 4:1)yielded 0.18 g (42%) of the sub-title compound.

[0363]¹H-NMR (500 MHz; CDCl₃): δ 7.04 (d, 1H); 6.76 (m, 2H); 3.79 (s,3H); 3.69 (s, 3H); 2.76 (m, 2H); 2.32 (m, 1H); 1.91 (m, 1H); 1.83 (m,2H); 1.75 (m, 1H); 1.58 (s, 3H)

[0364] (iii) 7-Methoxy-1-methyltetralin-1-yl-carboxylic Acid

[0365] A mixture of 7-methoxy-1-methyltetralin-1-yl-carboxylic acid,methyl ester (0.67 g; 2.9 mmol; from step (ii) above) and KOH (4 g) inEtOH:H₂0 (1:1; 50 mL) was stirred overnight. The resultant mixture wasdiluted with water and extracted with ether. The aqueous layer wasacidified (HCl) and extracted 3 times with ether. The combined organiclayer was washed with water, dried (Na₂SO₄), and concentrated. Yield0.58 g (81%).

[0366]¹H-NMR (300 MHz; CDCl₃): δ 7.02 (d, 1H); 6.85 (d, 1H); 6.75 (dd,1H); 3.77 (s, 3H); 2.75 (m, 2H); 2.32 (m, 1H); 1.91 (m, 1H); 1.82 (m,1H); 1.75 (m, 2H); 1.55 (s, 3H)

[0367] (iv) 7-Methoxy-1-methyltetralin-1-yl-C(O)-Aze-Pab(Z)

[0368] The sub-title compound was prepared according to the methoddescribed in Example 3(i) above from7-methoxy-1-methyltetralin-1-yl-carboxylic acid (0.14 g; 0.64 mmol; fromstep (iii) above), TBTU (0.31 g; 0.97 mmol), H-Aze-Pab(Z) (0.42 g; 0.97mmol) and DIPEA (0.50 g; 0.67 mmol). The crude product was purifiedusing flash chromatography (Si-gel, EtOAc). Yield 0.26 mg (72%).

[0369]¹H-NMR (400 MHz; CDCl₃): δ 8.40 (b, 0.5H, rotamer); 8.27 (b, 0.5H,rotamer); 7.90 (d, 2H); 4.84 (d, 2H); 7.45 (m, 2H); 7.4-7.25 (m, 5H);7.00 (t, 1h); 6.75 (dd, 0.5H, rotamer); 6.71 (dd, 0.5H, rotamer); 6.62(d, 0.5H, rotamer); 6.50 (d, 0.5H, rotamer); 5.22 (s, 2H); 4.87 (dd,1H); 4.65-4.40 (m, 2H); 3.78 (s, 3H); 3.69 (s, 3H); 3.60 (m, 1H); 2.78(m, 2H); 2.65 (m, 1H); 2.45 (m, 1H); 2.20 (m, 1H); 1.90 (m, 3H); 1.75(m, 3H); 1.50 (s, 3H)

[0370] (v) (R)- and (S)-7-Methoxy-1-methyltetralin-1-yl-C(O)-Aze-Pab

[0371] The title compounds were prepared according to the methoddescribed in Example 1(iv) above from7-methoxy-1-methyltetralin-1-yl-C(O)-Aze-Pab(Z) (0.10 g; 0.18 mmol; fromstep (iv) above) and Pd/C (10%) in EtOH (10 mL). The crude product waspurified using preparative RPLC (CH₃CN:0.1M ammonium acetate; 30:70 to32.5:67.5) yielding two diastereomers. The individual solutionscontaining the diastereomers were concentrated. Freeze-drying of thesolutions yielded the compound obtained from the fastest fraction(Compound 10A; 30 mg; 69%) and the slowest fraction (Compound 10B; 28mg; 64%).

[0372] Compound 10A (referred to hereinafter as (R) or (S)):

[0373] LC-MS (m/z) 435 (M+1)⁺ ¹H-NMR (400 MHz; D₂O): δ 7.74 (d, 2H);7.49 (d, 2H); 7.10 (d, 1H); 6.85 (dd, 1H); 6.66 (d, 1H); 4.53 (q, 1H);3.85 (m, 1H); 3.77 (s, 3H); 2.98 (m, 1H); 2.70 (m, 2H); 2.28 (m, 1H);2.03 (m, 2H); 1.95 (s, 3H); 1.88 (m, 1H); 1.72 (m, 1H); 1.44 (s, 3H)¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ 180.8;174.1; 167.6; 158.5

[0374] Compound 10B (referred to hereinafter as (S) or (R)):

[0375] LC-MS (m/z) 435 (M+1)⁺ ¹H-NMR (400 MHz; D₂O): δ 7.75 (d, 2H);7.50 (d, 2H); 7.06 (d, 1H); 6.80 (bd, 1H); 6.68 (b, 1H); 4.52 (q, 2H);3.75 (m, 4H); 2.88 (m, 1H); 2.68 (m, 2H); 2.37 (m, 1H); 1.90 (s, 3H);2.0-1.6 (m, 4H); 1.41 (s, 3H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/oramidine carbons) δ 180.9; 174.0; 167.5; 158.4

Example 11 4-Hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab×OAc

[0376] (i) 4-Hydroxy-6-methoxychroman-4-yl-carboxylic Acid Methyl Ester

[0377] The sub-title compound was prepared according to the methoddescribed by Bigge et al (J. Med. Chem. (1993) 36, 1977) from6-methoxychroman-4-one (1.29 g; 7.23 mmol), Me₃SiCN (0.79 g; 8.0 mmol)and ZnI₂ (20 mg; cat.). Yield 1.11 g (64%).

[0378]¹H-NMR (500 MHz; CD₃OD): δ 6.80 (dd, 1H); 6.73 (d, 1H); 6.72 (s,1H); 4.28 (m, 1H); 4.14 (dt, 2H); 3.74 (s, 3H); 3.70 (s, 3H); 2.47 (m,1H); 2.02 (m, 1H)

[0379] (ii) 4-Hydroxy-6-methoxychroman-4-yl-carboxylic Acid

[0380] The sub-title compound was prepared according to the methoddescribed in Example 1(ii) above from4-hydroxy-6-methoxychroman-4-yl-carboxylic acid, methyl ester (1.09 g;4.58 mmol; from step (i) above) and LiOH.H₂O (0.39 g; 9.2 mmol). Yield0.71 g (69%).

[0381] LC-MS (m/z) 223 (M+1)⁺ ¹H-NMR (300 MHz; CD₃OD): δ 6.81 (m, 1H);6.77 (m, 1H); 6.74 (m, 1H); 4.31 (m, 1H); 4.14 (m, 1H); 3.71 (s, 3H);2.50 (m, 1H); 2.03 (m, 1H)

[0382] (iii) 4-Hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab(Z)

[0383] The sub-title compound was prepared according to the methoddescribed in Example 3(i) above from4-hydroxy-6-methoxychroman-4-yl-carboxylic acid (0.104 g; 0.464 mmol;from step (ii) above), TBTU (0.29 g; 0.90 mmol), DMF (8 mL), DIPEA (80μL+320 μL; 0.46 mmol+1.84 mmol) and H-Aze-Pab(Z) (0.4 g; 0.91 mmol). Thecrude product, 0.27 g of a yellow viscous oil, was purified usingpreparative RPLC (CH₃CN:0.1M ammonium acetate 40:60), the fractions ofinterest were concentrated and extracted with EtOAc. Yield 0.089 g(33%).

[0384] LC-MS (m/z) 573 (M+1)⁺

[0385] (iv) 4-Hydroxy-6-methoxychroman-4yl-C(O)-Aze-Pab×HOAc

[0386] The title compound was prepared according to the method describedin Example 1(iv) above from4-hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab(Z) (0.089 g; 0.155 mmol;from step (iii) above), AcOH (0.25 μL, 0.44 mmol) and Pd/C (5%; 0.089g). Yield 55.5 mg (72%).

[0387] LC-MS (m/z) 439 (M+1)⁺

[0388]¹H-NMR (300 MHz; CD₃OD): (complex due todiastereomerism/rotamerism) δ 7.7-7.5 (m, 2H); 7.5-7.3 (m, 2H);6.75-6.55 (m, 3H); 4.8 (m, 1H, partly hidden by HDO); 4.5-3.5 (m, 9H,thereof 3.74 (s) and 3.69 (s); 2.7-1.7 (m, 7H; thereof 1.95, s, 3H)¹³C-NMR (75 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ 176.4;173.1; 168.1

Example 12 (S)- and (R)-1-Hydroxy-4-methoxyindan-1-yl-C(O)-Aze-Pab

[0389] (i) 4-Methoxy-1-indanone

[0390] Cs₂CO₃ (7 g; 21.5 mmol) followed by CH₃I (10 g; 70 mmol) wasadded to a solution of 4-hydroxy-1-indanone (5.0 g; 34 mmol) in THF (30mL) and the mixture was stirred at RT for 60 h. The reaction mixture wasfiltered and concentrated, and the crude product was purified usingflash chromatography (SiO₂; methylene chloride) to yield 3.1 g (56%) ofthe sub-title substance.

[0391] (ii) 1-Hydroxy-4-methoxyindan-1-yl-carboxylic Acid Ethyl Ester

[0392] The sub-title compound was prepared according to a methoddescribed by Bigge et al (J. Med. Chem. (1993) 36, 1977) from4-methoxy-1-indanone (2.2 g; 13.5 mmol; from step (i) above), Me₃SiCN(2.0 g; 20 mmol), and ZnI₂ (200 mg; 0.62 mmol; cat.). Yield 0.9 g (28%).

[0393]¹H-NMR (400 MHz; CDCl₃): δ 7.21 (t, 1H); 6.85 (d, 1H); 6.79 (d,1H); 4.20 (m, 2H); 3.85 (s, 3H); 3.07 (m, 3H); 2.97 (m, 1H); 2.67 (m,1H); 2.27 (m, 1H); 1.20 (t, 3H)

[0394] (iii) 1-Hydroxy-4-methoxyindan-1-yl-carboxylic Acid

[0395] NaOH (19M; 1.0 mL) was added to a solution of1-hydroxy-4-methoxyindan-1-yl-carboxylic acid ethyl ester (0.90 g; 3.8mmol; from step (ii) above) in EtOH (20 mL) and the solution was stirredfor 30 minutes. Brine (40 mL) was added and the mixture was washed withEtOAc, cautiously made acidic to pH 2 (HCl; 2M), and the aqueoussolution was extracted with EtOAc. The organic layer was washed withbrine, dried (Na₂SO₄) and concentrated, yielding 0.70 g (88%) of thesub-title substance.

[0396]¹H-NMR (400 MHz; CDCl₃): δ 7.23 (t, 1H); 6.89 (d, 1H); 6.81 (d,1H); 3.85 is (s, 3H); 3.0 (m, 2H); 2.77 (m, 1H); 2.3 (m, 1H)

[0397] (iv) 1-Hydroxy-4-methoxyindan-1-yl-C(O)-Aze-Pab(Z)

[0398] The sub-title compound was prepared according to the methoddescribed in Example 4(iv) above from1-hydroxy-4-methoxyindan-1-yl-carboxylic acid (350 mg; 1.68 mmol; fromstep (iii) above), methylene chloride (25 mL), H-Aze-Pab(Z) (750 mg; 1.7mmol), TBTU (600 mg; 1.8 mmol) and DIPEA (770 mg; 1.8 mmol). The mixturewas concentrated, and the remainder was purified using flashchromatography (Si-gel; acetone:EtOAc), yielding 350 mg (37.5%).

[0399]¹H-NMR (400 MHz; CDCl₃) (complex due to diastereomers/rotamers) δ8.05 (t, 0.5H, rotamer); 7.95 (t, 0.5H, rotamer); 7.82 (dd, 2H); 7.45(d, 2H); 7.35 (m, 5H); 7.20 (m, 1H); 6.82 (m, 2H); 4.92 (m, 1H); 4.48(m, 2H); 3.84 (s, 3H); 3.66 (m, 2H); 3.30 (m, 1H); 2.95 (m, 1H); 2.55(m, 1H); 2.46 (m, 1H); 2.30 (m, 1H)

[0400] (v) (S)- and (R)-1-Hydroxy-4-methoxyindan-1-yl-C(O)-Aze-Pab

[0401] Ammonium formate (1.0 g; 16 mmol) and Pd/C (5%; 200 mg) wereadded to a solution of 1-hydroxy-4-methoxyindan-1-yl-C(O)-Aze-Pab(Z)(340 mg; 0.61 mol; from step (iv) above) in MeOH (30 mL). Formic acid(200 mg; 4.4 mmol) was added and the mixture was stirred for 30 minutes.The reaction mixture was filtered through Hyflo and the solution wasconcentrated. The crude product was purified using preparative RPLC(CH₃CN:0.1M ammonium acetate; 15:85). The fractions of interest werecollected and concentrated, and the water solution was freeze dried,yielding a faster moving fraction (Compound 12A; 50 mg; 34%) and aslower moving fraction (Compound 12B; 5 mg; 3.4%).

[0402] Compound 12A (referred to hereinafter as (R) or (S)):

[0403] LC-MS (m/z) 423 (M+1)⁺ ¹H-NMR (400 MHz; CD₃OD): (complex due torotamerism) δ 7.74 (d, 2H, minor rotamer); 7.70 (d, 2H, major rotamer);7.60 (d, 2H, minor rotamer); 7.48 (d, 1H, major rotamer); 7.20 (t, 1H);6.95 (d, 1H, major rotamer); 6.87 (d, 1H, minor rotamer); 6.84 (d, 1H,major rotamer); 6.83 (d, 1H, minor rotamer); 4.82 (m, 1H); 4.5 (m, 1H);4.6-4.4 (m, 2H); 4.11 (m, 1H, major rotamer); 4.00 (m, 1H, minorrotamer); 3.82 (s, 3H); 3.0 (m, 1H); 2.9 (m, 1H); 2.65 (m, 1H), 2.5 (m,1H); 2.3-2.0 (m, 2H) ¹³C-NMR (100 MHz; CD₃OD): (carbonyl and/or amidinecarbons) δ 180.3; 176,4; 173.0; 167.9

[0404] Compound 12B (referred to hereinafter as (5) or (R)):

[0405] LC-MS (m/z) 423 (M+1)⁺ ¹H-NMR (400 MHz; CD₃OD): (complex due torotamerism) δ 7.8-7.7 (m, 2H); 7.54 (d, 2H); 7.23 (m, 1H); 6.97 (m, 1H);6.9-6.8 (m, 1H); 4.80 (m, 1H); 4.6-4.4 (m, 2H); 4.25 (m, 1H); 4.1-3.9(m, 1H); 3.82 (s, 3H); 3.0-2.85 (m, 2H); 2.8-2.5 (m, 2H); 2.3-2.1 (m,2H); 1.90 (s, 3H)

Example 13 1-Hydroxy-5-methoxytetralin-1-yl-C(O)-Aze-Pab(OH)

[0406] A solution of hydroxylamine×HCl (39 mg; 0.56 mmol) and TEA (0.26mL; 1.86 mmol) in THF (10 mL) was sonicated at 40° C. for 1 h,whereafter 1-hydroxy-5-methoxytetralin-1-yl-C(O)-Aze-Pab(Z) (53 mg;0.093 mmol; see Example 5(iii) above) dissolved in a small amount of THFwas added, and the mixture was stirred at 40° C. for 3 days. The mixturewas concentrated, and the product was purified using preparative RPLC(CH₃CN:0.1M ammonium acetate; 30:70). The fractions of interest wereconcentrated and the remainder was freeze dried. Yield 29 mg (70%).

[0407] LC-MS (m/z) 453 (M+1)⁺ ¹H-NMR (400 MHz; CD₃OD): (complex due todiastereomers/rotamers) δ 7.58 (m, 2H); 7.33 (dd, 2H); 7.16 (m, 1H);7.85 (m, 2H); 4.78 (dd, 2H); 4.44 (m, 2H); 4.2-4.0 (m, 2H); 3.80 (s,3H); 3.58 (m, 0.5H, rotamer); 3.47 (m, 0.5H, rotamer); 2.91 (bd, 1H);2.44 (m, 2H); 2.34 (m, 1H); 2.19 (m, 1H); 2.08 (m, 2H); 1.98 (b, 2H);1.89 (b, 2H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidine carbons)δ 178.0; 177.8; 172.9, 158.4; 158.2; 155.3

Example 14 (S)- or (R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(OH)

[0408] The title compound was prepared according to the method describedin Example 13 above from hydroxylamine×HCl (48 mg; 0.69 mmol), TEA (0.32mL; 2.31 mmol) and (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Z) (66 mg; 0.12 mmol;Compound 3A from Example 3(i) above). The mixture was concentrated, andthe crude product was purified using preparative RPLC (CH₃CN:0.1Mammonium acetate; 28:72) yielding 17 mg (31%). Purity 94.5%,diastereomeric ratio 87:13.

[0409] LC-MS (m/z) 453 (M+1)⁺ ¹H-NMR (400 MHz; CD₃OD): δ 5.75 (d, 2H);7.37 (m, 3H); 7.04 (d, 1H); 6.81 (m, 1H); 4.82 (m, 1H); 4.44 (m, 2H);4.28 (m, 1H); 4.08 (m, 1H); 3.72 (s, 3H); 3.64 (m, 1H); 2.72 (m, 3H);2.40 (m, 1H); 2.22 (m, 1H); 2.12 (m, 2H); 1.95 (m, 2H); 1.88 (m, 3H)¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ 177.6;172.6, 159.4

Example 15 4-Hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab(OH)

[0410] The title compound was prepared according to the method describedin Example 13 above from hydroxylamine×HCl (74 mg; 1.06 mmol), TEA (0.50mL; 3.6 mmol) and 4-hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab(Z) (92mg; 0.16 mmol; see Example 1 1(iii) above). The crude product waspurified using preparative RPLC (CH₃CN:0.1M ammonium acetate; 28:72)yielding 55 mg (75%). Diastereomeric ratio 53:47.

[0411]¹H-NMR (400 MHz; CDCl₃): (complex due todiastereomerism/rotamerism) δ 7.65-7.5 (m, 2H); 7.4-7.3 (m, 2H);6.85-6.65 (m, 3H); 4.81 (m, 1H, partly hidden by HDO); 4.5-3.9 (m, 5H);3.9-3.6 (m, 4H); 2.8-1.9 (m, 4H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyland/or amidine carbons) δ 176.5; 176.2; 172.8; 155.2

Example 16 4-Hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab(OMe)

[0412] The title compound was prepared according to the method describedin Example 3(i) above from 4-hydroxy-6-methoxychroman-4-yl-carboxylicacid (95 mg; 0.42 mmol; see Example 11(ii) above), TBTU (0.26 g; 0.81mmol), DMF (5 mL), H-Aze-Pab(OMe)×HCl (0.256 g; 0.81 mmol; see Example4(iii) above) and DIPEA (75+300 μL; 0.42+1.68 mmol). The crude productwas purified using preparative RPLC (CH₃CN:0.1M ammonium acetate;30:70), yielding 67 mg (37%).

[0413]¹H-NMR (400 MHz; CDCl₃): (complex due todiastereomerism/rotamerism) δ 7.65-7.5 (m, 2H); 7.4-7.3 (m, 2H);6.85-6.7 (m, 3H); 4.80 (m, 1H, hidden by HDO); 4.5-4.0 (m, 5H); 3.81 (s,3H); 3.75-3.65 (m, 4H); 2.8-1.9 (m, 4H) ¹³C-NMR (100 MHz; CDCl₃):(carbonyl and/or amidine carbons; complex due todiastereomerism/rotamerism) δ 177.8; 176.5; 176.1; 172.8; 172.6; 155.2;155.0

Example 17 (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(C(O)OCH₂CCl₃)

[0414] NaOH (aq; 2M; 0.78 mL), and then 2,2,2-trichloroethylchloroformate (21 μL; 0.155 mmol), were added to an ice-cold solution of(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab×HOAc (70 mg;0.14 mmol; see Example 3 above) in THF (3 mL), and the mixture wasstirred for 3 hours. The reaction mixture was diluted in water and theresultant mixture was extracted 4 times with methylene chloride. Thecollected organic phase was washed with brine, dried (Na₂SO₄) andevaporated. Yield 79.8 mg (92.5%).

[0415] LC-MS (m/z) 613 (M+1)⁺ ¹H-NMR (400 MHz; CDCl₃): δ 9.42 (b, 1H);7.98 (t, 1H); 7.83 (d, 2H); 7.30 (b, 1H); 7.29 (d, 2H); 7.06 (d, 1H);6.84 (dd, 1H); 6.67 (d, 1H); 4.92 (dd, 1H); 4.86 (s, 2H); 4.48 (m, 2H);4.12 (s, 1H); 3.86 (m, 1H); 3.75 (s, 3H); 3.08 (m, 1H); 2.81 (db, 1H);2.58 (m, 2H); 2.27 (m, 1H); 1.95 (m, 3H) ¹³C-NMR (100 MHz; CDCl₃):(carbonyl and/or amidine carbons) δ 178.7; 171.5; 170.1; 164.0

Example 18 (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(C(O)OCH₂CH₃)

[0416] The title compound was prepared according to the method describedin Example 17 above from (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab×HOAc (52 mg; 0.10mmol; see Example 3 above), NaOH (aq; 2M; 0.58 mL), and ethylchloroformate (9.4 μL; 0.089 mmol). The crude product was purified usingpreparative RPLC (CH₃CN:0.1M ammonium acetate 30:70). Yield 29 mg (69%).

[0417] LC-MS (m/z) 509 (M+1)⁺ ¹H-NMR (400 MHz; CDCl₃): δ 9.55 (b, 1H);7.96 (t, 1H); 7.85 (d, 2H); 7.34 (d, 2H); 7.06 (d, 1H); 6.83 (dd, 1H);6.68 (d, 1H); 4.94 (dd, 1H); 452 (m, 3H); 4.24 (q, 2H); 3.84 (m, 1H);3.77 (s, 3H); 3.04 (m, 1H); 2.82 (m, 1H); 2.62 (m, 2H); 2.27 (m, 1H);2.0-1.85 (m, 5H); 1.37 (t, 3H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyland/or amidine carbons) δ 178.9; 171.4. 159.6

Example 19 7-Methoxy-1-allyltetralin-1-yl-C(O)-Aze-Pab×HOAc

[0418] (i) 7-Methoxy-1-allyltetralin-1-yl-carboxylic Acid

[0419] The sub-title compound was prepared according to the methoddescribed in Example 10(ii) above from 7-methoxytetralin-1-yl-carboxylicacid, methyl ester (0.80 g; 3.6 mmol; see Example 10(i) above), NaH (55%in oil; 0.23 mg; 5.4 mmol), and allyl bromide (0.65 g, 5.4 mmol),whereafter the crude product was hydrolysed directly according to themethod described in Example 10(iii) above with KOH (3 g) in EtOH:H₂O (40mL; 1:1). Yield 0.39 g (44%).

[0420]¹H-NMR (400 MHz; CDCl₃): δ 7.00 (d, 1H); 6.93 (d, 1H); 6.72 (dd,1H); 5.64 (m, 1H); 5.05 (m, 2H); 3.75 (s, 3H); 2.85-2.60 (m, 4H); 2.20(m, 2H); 1.95-1.70 (m, 3H)

[0421] (ii) Boc-Aze-Pab×HCOOH

[0422] The sub-title compound was prepared according to the methoddescribed in Example 12(v) above from ammonium formate (3.0 g; 50 mmol),Pd/C (5%; 1.0 g), Boc-Aze-Pab(Z) (4.7 g; 10 mmol; see internationalpatent application WO 94/29336) and formic acid (1.0 g; 22 mmol) in 50mL of MeOH. The crude product was suspended in CH₂Cl₂ (50 mL), filteredand washed with more CH₂Cl₂. The solid material was dried and used inthe following step without further purification.

[0423] (iii) Boc-Aze-Pab(Teoc)

[0424] Teoc-p-nitrophenyl carbonate (3.5 g; 12.3 mmol) was added to asolution of Boc-Aze-Pab×HCOOH (3.7 g; 10 mmol; from step (ii) above) inTHF (100 mL) whereafter a solution of K₂CO₃ (1.8 g; 13 mmol) in water(20 mL) was added over 2 minutes. The resultant solution was stirred for3 days, concentrated, and the remainder was taken up in EtOAc (150 mL)and NaOH (aq; 0.5M; 50 mL). The organic layer was washed with brine(2×50 mL), dried (Na₂SO₄) and concentrated. The crude product waspurified using flash chromatography (Si-gel; methylene chloride:acetone;4:1). Yield 4.6 g (96%).

[0425]¹H-NMR (500 MHz; CDCl₃): δ 7.86 (d, 2H); 7.39 (d, 2H); 4.72 (bt,1H); 4.53 (b, 2H); 3.93 (q, 1H); 3.81 (q, 1H); 2.48 (b, 2H); 1.43 (s,9H)

[0426] (iv) H-Aze-Pab(Teoc)×HCl

[0427] A solution of Boc-Aze-Pab(Teoc) (4.6 g; 9.6 mmol; from step (ii)above) in methylene chloride (150 mL) was saturated with dry HCl. Thesolution was kept at RT in a stoppered flask for 10 minutes, whereafterit was concentrated. Yield 4.2 g (97%).

[0428]¹H-NMR (400 MHz; CD₃OD): δ 7.80 (d, 2H); 7.60 (d, 2H); 5.10 (t,1H); 4.60 (s, 2H); 4.15 (q, 1H); 3.97 (q, 1H); 2.86 (m, 1H); 2.57 (m,1H)

[0429] (v) 7-Methoxy-1-allyltetralin-1-yl-C(O)-Aze-Pab(Teoc)

[0430] The sub-title compound was prepared according to the methoddescribed in Example 3(i) above from7-methoxy-1-allyltetralin-1-yl-carboxylic acid (0.30 g; 1.2 mmol; fromstep (i) above), TBTU (0.43 g; 1.3 mmol), H-Aze-Pab(Teoc) (0.60 g; 1.3mmol; from step (iv) above) and DIPEA (0.69 g; 5.4 mmol). The crudeproduct was purified using flash chromatography (Si-gel; EtOAc). Yield0.41 mg (56 %).

[0431]¹H-NMR (500 MHz; CDCl₃): (complex due todiastereomerism/rotamerism) δ 8.35 (b, 0.5H); 8.20 (bt, 0.5H); 7.90 (d,1H); 7.85 (d, 1H); 7.90 (d, 1H); 7.35 (d, 1H); 7.01 (t, 1H); 6.75 (m,1H); 6.65 (d, 0.5H); 6.53 (d, 0.5H); 5.80-5.65 (m, 1H); 5.02 (dd, 1H);4.96 (m, 1H); 4.87 (dd, 1H); 4.61 (m, 1H); 4.43 (dt, 1H); 4.25 (m, 2H);3.70 (m+s, 3H); 3.54 (m, 0.5H); 2.95-2.40 (m, 6H); 2.23 (m, 1H); 2.13(m, 1H); 1.98 (m, 2H); 1.80 (m, 2H); 1.13 (m, 2H); 0.13 (d, 9H)

[0432] (vi) 7-Methoxy-1-allyltetralin-1-yl-C(O)-Aze-Pab×HOAc

[0433] A solution of Bu₄NF (1M in THF; 0.66 mL) was added to a solutionof 7-methoxy-1-allyltetralin-1-yl-C(O)-Aze-Pab(Teoc) (0.36 g; 0.60 mmol;from step (v) above) in THF (40 mL), and the solution was stirred at 60°C. for 24 h. The crude product was purified using preparative RPLC(CH₃CN:0.1M ammonium acetate (50:50)) and freeze dried. Yield 0.22 g(71%).

[0434]¹H-NMR (500 MHz; CDCl₃): δ 7.77 (dd, 2H); 7.52 (t, 2H); 7.13 (t,1H); 6.87 (dt, 1H); 6.77 (dd, 1H); 5.71 (m,1H); 5.02 (m, 2H); 4.53 (b,1H); 3.85-3.65 (m, 4H); 3.02 (m, 1H); 2.70 (b, 4H); 2.40-2.20 (m, 1H);2.05-1.70 (b, 8H; thereof 1.92; s) ¹³C-NMR (100 MHz; D₂O): (carbonyland/or amidine carbons) δ 179.1; 173.7; 1i7.3; 158.5

[0435] LC-MS (m/z) 459 (M−1)⁻

Example 20 (S)- or (R)-1-Hydroxy-7-chlorotetralin-1-yl-C(O)-Aze-Pab

[0436] (i) 7-Amino-1-tetralone

[0437] Ammonium formate (2 g), Pd/C (5%; 1 g), and formic acid (0.5 g;cat.) were added in that order to a solution of 7-nitro-1-tetralone(1.95 g; 10 mmol) in methanol (50 mL), and the mixture was stirred for30 minutes. The solution was filtered, and the filtrate wasconcentrated. The remainder was soaked with methylene chloride (50+25mL), and the mixture was filtered and concentrated. Yield 1.4 g (88%).

[0438]¹H-NMR (500 MHz; CDCl₃): δ 7.32 (d, 1H); 7.03 (d, 1H); 6.83 (dd,1H); 3.70 (b, 2H); 2.85 (t, 2H); 2.61 (t, 2H); 2.10 (m, 2H)

[0439] (ii) 7-Chloro-1-tetralone

[0440] NaNO₂ (0.7 g; 10 mmol) dissolved in water (10 mL) was added withstirring to an ice-cold solution of 7-amino-1-tetralone (1.4 g; 8.8mmol; from step (i) above) in conc HCl (aq.) over a period of 5 minutes.The resultant cold solution was then added slowly to an ice-coldsolution of CuCl (1.5 g, 15 mmol) in conc. HCl (aq.), whereafter theresultant solution was stirred at RT for 2 hours and at 60° C. for 30minutes. The slurry was cooled with ice, and the resultant precipitatewas suction filtered, washed with water, and air dried. Yield 1.50 g(94%).

[0441]¹H-NMR (500 MHz; CDCl₃): δ 8.00 (d, 1H); 7.41 (dd, 1H); 7.20 (d,1H); 2.95 (t, 2H); 2.66 (m, 2H); 2.14 (m, 2H)

[0442] (iii) 7-Chloro-1-hydroxytetralin-1-carboxylic Acid Ethyl Ester

[0443] Prepared according to the method described by C. F. Bigge et alin J. Med. Chem (1993) 36, 1977 using 7-chloro-1-tetralone (1.5 g; 8.3mmol; from step (ii) above), Me₃Si—CN (1.0 g; 10 mmol), and ZnI₂ (0.3g). Yield 0.8 g (36%).

[0444]¹H-NMR (600 MHz; CDCl₃): δ 7.72 (s, 1H); 7.25 (d, 1H); 7.17 (d,1H); 7.09 (dd, 1H); 4.35-4.20 (m, 2H); 2.80 (m, 2H); 2.35 (m, 1H);2.12-1.92 (m, 3H), 1.25 (m, 3H)

[0445] (iv) 7-Chloro-1-hydroxytetraline-1-carboxylic Acid

[0446] NaOH (10M, 1 mL) was added to a solution of7-chloro-1-hydroxytetraline-1-carboxylic acid, ethyl ester (0.8 g; 3.1mmol; from step (iii) above) in DMSO (20 mL), and the mixture was heatedto 100° C. for 3 hours. The resultant mixture was diluted with crushedice (40 g) and brine (40 mL), and the mixture was extracted with EtOAc.The aqueous layer was acidified to pH 2 with 2M HCl and extracted withEtOAc (2×40 mL). The combined organic layer was dried (Na₂SO₄) andconcentrated. Yield 0.22 mg (30%).

[0447]¹H-NMR (500 MHz; CDCl₃): δ 7.25 (d, 1H); 7.20 (d, 1H); 7.09 (d,1H); 2.80 (m, 2H); 2.27 (m, 1H); 2.17-2.00 (m, 2H); 1.98 (m, 1H)

[0448] (v) 7-Chloro-1-hydroxytetralin-1-1-C(O)-Aze-Pab(Z)

[0449] HATU (400 mg; 1.05 mmol) was added to a solution of7-chloro-1-hydroxytetraline-1-carboxylic acid (220 mg; ca 1 mmol; fromstep (iv) above) in DMF (50 mL) and, after stirring for a short time, asolution of H-Aze-Pab(Z)×2HCl (450 mg; 1.02 mmol) and2,4,6-trimethylpyridine (425 mg, 3.5 mmol) in DMF (10 mL) was addeddropwise. After stirring overnight, the resultant mixture was dilutedwith an aqueous solution of NaCl (15%; 100 mL) and extracted with EtOAc(2×50 mL). The organic layer was washed with brine (20 mL), dried(Na₂SO₄), and concentrated. The remainder was purified using flashchromatography (Si-gel; EtOAc). Yield 300 mg (52%).

[0450]¹H-NMR (400 MHz; CDCl₃): (complex due to diastereomerism and/orrotamerism) δ 7.89 (d, 1H); 7.82 (d, 1H); 7.42 (d, 2H); 7.40-7.30 (m,6H); 7.18 (m, 1H); 7.06 (d, 1H); 5.20 (s, 2H); 4.93 (m, 1H); 4.60-4.40(m, 3H); 3.83 (m, 0.5H); 3.72 (m, 0.5H); 3.07 (m, 1H); 2.7-2.5 (m, 3H);2.40 (m, 1H); 2.03-1.80 (m, 5H)

[0451] (vi) 7-Chloro-1-hydroxytetralin-1-yl-C(O)-Aze-Pab×HOAc

[0452] Anisol (65 mg; 0.6 mmol) and trifluoromethanesulfonic acid (400mg; 2.6 mmol) were added, in that order, to a solution of7-chloro-1-hydroxytetralin-1-yl-C(O)-Aze-Pab(Z) (300 mg; 0.52 mmol; fromstep (v) above) in methylene chloride (20 mL) and the solution wasstirred at RT for 10 minutes. Water (20 mL) was added and the organicphase was separated and removed, whereafter the aqueous phase wasadjusted to pH 4-5 with saturated NaHCO₃ (aq). The solution waspartially concentrated and the crude product was purified usingpreparative RPLC (CH₃CN:water; 10:90 to 90:10). The fractions ofinterest were partially concentrated, a few drops of HOAc (conc.) wereadded, and the solution was freeze dried. Yield 40 mg (15%).

[0453]¹H-NMR (500 MHz; CD₃OD): δ 7.78 (dd, 2H); 7.59 (m, 2H); 7.30 (d,1h); 7.22 (d, 1H); 7.15 (d, 1H); 4.65-4.35 (m, 3H); 4.20-3.90 (m, 1H);2.85-2.70 (m, 1H); 2.55 (m, 1H); 2.35-1.95 (m, 9H) ¹³C-NMR (100 MHz;CDCl₃): (carbonyl and/or amidine carbons) δ 177.0; 172.8; 167.9

Example 21 1-n-Propyl-7-methoxytetralin-1-yl-C(O)-Aze-Pab×HOAc

[0454] A small amount of Pd/C (10%) was added to a solution of1-allyl-7-methoxytetralin-1-yl-C(O)-Aze-Pab×HOAc (80 mg; 0.15 mmol; seeExample 19 above) in EtOH (5 mL) and the mixture was hydrogenated atambient temperature and pressure for 2 h. The mixture was filteredthrough Celite and the resultant solution was concentrated. Freezedrying from water yielded 68 mg (85%) of the title compound.

[0455]¹H-NMR (400 MHz; CDCl₃): δ 7.77 (t, 2H); 7.52 (t, 2H); 7.12 (t,1H); 6.87 (m, 1H); 6.75 (d, 1H); 4.75 (m, 1H; partially hidden); 4.54(s, 2H); 3.77 (m, 4H); 3.66 (m, 1H); 3.10 (m, 1H); 2.70 (b, 2H); 2.30(m, 1H); 2.1-1.6 (m, 10H; thereof 1.91, s, 3H); 1.25 (m, 1H); 1.10 (m,1H); 0.83 (q, 3H) LC-MS (m/z) 463 (M+1)⁺

Example 22 6-Chloro-4-hydroxychroman-4-yl-C(O)-Aze-Pab×HOAc

[0456] (i) 6-Chloro4-hydroxychroman-4-yl-carboxylic Acid, Methyl Ester

[0457] The sub-title compound was prepared according to the methoddescribed by Bigge et al (J. Med. Chem (1993) 36, 1977ff) from6-chlorochromanone (2.45 g; 13.4 mmol), Me₃SiCN (1.51 g; 15.2 mmol), andZnI₂ (40 mg; cat.). Yield 0.58 g (18%).

[0458]¹H-NMR (300 MHz; CDCl₃): δ 7.17 (d, 1H); 7.08 (d, 1H); 6.82 (d,1H); 4.41 (m, 1H); 4.37 (m, 1H); 2.47 (m, 1H); 2.09 (m, 1H)

[0459] (ii) 6-Chloro-4-hydroxychroman-4-yl-carboxylic Acid

[0460] LiOH.H₂O (0.19 g; 4.6 mmol) and water (4 mL) were added to asolution of 6-chloro-4-hydroxychroman-4-yl-carboxylic acid, methyl ester(0.56 g, 2.3 mmol; from step (i) above) in THF (6 mL). The reactionmixture was stirred at room temperature for 3 h, THF was evaporated andthe water solution was washed with methylene chloride. The reactionmixture was acidified with HCl (2M) and extracted with ethyl acetate.The organic layer was dried (Na₂SO₄) and evaporated, yielding a slowlycrystallizing oil. Yield: 490 mg (93%).

[0461] LC-MS (m/z) 228 (M−1)⁻

[0462] (iii) 6-Chloro-4-hydroxychroman-4-yl-C(O)-Aze-Pab(Teoc)

[0463] A solution of 6-chloro-4-hydroxychroman-4-yl-carboxylic acid (222mg; 1.00 mmol; from step (ii) above) and HATU (370 mg, 0.97 mmol) in DMF(5 mL) was stirred at 0° C. for 1.5 h, and a mixture ofH-Aze-Pab(Teoc)×HCl (440 mg; 0.98 mmol; see Example 19(iv) above) and2,4,6-trimethylpyridine (0.48 g; 3.9 mmol) in DMF (5 mL) was added at 0°C. After stirring for 3 h at 0° C., the reaction mixture wasconcentrated, and the crude product was purified using preparative RPLC(CH₃CN:0.1M ammonium acetate (55:45)). The fractions of interest werepartly concentrated and extracted with methylene chloride. The organiclayer was dried (Na₂SO₄) and concentrated yielding 350 mg (67%) of adiastereomeric mixture.

[0464]¹H-NMR (400 MHz; CDCl₃): δ 7.31 (m, 1H); 7.19 (dt, 1H); 7.09 (d,0.5H); 7.00 (d, 0.5H); 6.88 (dd, 1H); 4.93 (m, 1H); 4.80 (br, 0.5H);4.61 (dd, 1H); 4.53-4.43 (m, 2H); 4.36 (m, 1H); 4.15 (t, 1H); 3.89 (m,0.5H); 3.74 (m, 0.5H); 3.09 n(m, 1H); 2.46-2.28 (m, 1H); 2.21 (m, 1H);1.96 (m, 1H); 0.06 (s, 9H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/oramidine carbons) δ 176.9; 171.5; 171.3; 169.8; 155.4; 155.2 LC-MS (m/z)588 (M+1)⁺

[0465] (iv) 6-Chloro-4-hydroxychroman-4-yl-C(O)-Aze-Pab×HOAc

[0466] Bu₄NF (1.0M in THF; 0.35 mL) was added to a solution of6-chloro-4-hydroxychroman-4-yl-C(O)-Aze-Pab(Teoc) (190 mg; 0.32 mmol;from step (iii) above) in THF (20 mL) at 0° C. The solution was stirredfor two days at 40° C. The solution was concentrated and the crudematerial was purified using preparative RPLC (CH₃CN:0.1M ammoniumacetate (25:75)). Yield 115 mg (71%).

[0467]¹H-NMR (400 MHz; CD₃OD): δ 7.73 (m, 2H); 7.55 (m, 2H); 7.28 (dd,1H); 7.15 (m, 1H); 6.79 (m, 1H); 4.60 (m, 1H); 4.47 (m, 2H); 4.33 (m,1H); 4.15 (m, 2H); 2.8-2.46 (m, 1H); 2.38 (m, 1H); 2.23 (m, 1H); 2.06(m, 1H); 1.90 (s, 3H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidinecarbons) δ 175.9; 175.6; 174.4; 173.1; 173.0 LC-MS (m/z) 444 (M+1)⁺

Example 23 4-Hydroxychroman-4-yl-C(O)-Aze-Pab×HOAc

[0468] Pd/C (5%; 25 mg) was added to a solution of6-chloro-4-hydroxychroman-4-yl-C(O)-Aze-Pab×HOAc (14.7 mg; 0.029 mmol;see Example 22 above) in EtOH (5 mL), and the mixture was hydrogenatedat ambient temperature and pressure for one day. The mixture wasfiltered through Celite, and the crude product was purified usingpreparative RPLC (CH₃CN:0.1M ammonium acetate (25:75)). The fractions ofinterest were concentrated. Freeze drying yielded 4 mg (30%) of thetitle compound.

[0469]¹H-NMR (400 MHz; CD₃OD): δ 7.79 (m, 2H); 7.56 (m, 2H); 7.4-7.2 (m,2H); 7.00 (m, 2H); 4.96 (dd, 1H); 4.5-4.3 (m, 2H); 4.20 (m, 2H); 3.88(m, 1H); 2.8-2.4 (m, 2H); 2.27 (m, 1H); 2.17 (m, 1H); 2.07 (s, 3H)¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ 173.7;167.7 LC-MS (m/z) 409 (M+1)⁺

Example 24 6,8-Dichloro-4-hydroxychroman-4-yl-C(O)-Aze-Pab×HOAc

[0470] (i) 6,8-Dichloro-4-hydroxychroman-4-yl-carboxylic Acid, MethylEster

[0471] The sub-title compound was prepared according to the methoddescribed by Bigge et al (J. Med. Chem. (1993) 36, 1977ff) from6,8-dichlorochromanone (1.36 g; 6.27 mmol), Me₃SiCN (0.68 g; 6.9 mmol),and ZnI₂ (20 mg; cat.). Yield 0.52 g (30%).

[0472]¹H-NMR (300 MHz; CDCl₃): δ 7.30 (s, 1H); 7.00 (s, 1H); 4.53 (m,1H); 4.33 (m, 1H); 3.83 (s, 3H); 2.47 (m, 1H); 2.12 (m, 1H)

[0473] (ii) 6,8-Dichloro-4-hydroxychroman-4yl-carboxylic Acid

[0474] LiOH.H₂O (0.15 g; 3.6 mmol) and water (2 mL) were added to asolution of 6,8-dichloro-4hydroxychroman-4yl-carboxylic acid, methylester (0.50 g; 1.8 mmol; from step (i) above) in THF (5 mL). Theresultant mixture was stirred at room temperature for 30 min., THF wasevaporated and the water phase was washed with methylene chloride. Thereaction mixture was made acidic with HCl (2M) and extracted withmethylene chloride. The organic layer was dried (Na₂SO₄) and evaporated,yielding the sub-title compound. Yield: 390 mg (83%).

[0475] LC-MS (m/z) 262 (M−1)⁻

[0476] (iii) 6,8-Dichloro-4-hydroxychroman-4-yl-C(O)-Aze-Pab(Teoc)

[0477] The sub-title compound was prepared according to a methoddescribed in Example 22(iii) above from6,8-dichloro-4-hydroxychroman-4-yl-carboxylic acid (100 mg; 0.38 mmol;from step (ii) above), HATU (160 mg; 0.42 mmol), H-Aze-Pab(Teoc)×HCl,(190 mg; 0.42 mmol; see Example 19(iv) above), and2,4,6-trimethylpyridine (0.19 g; 1.6 mmol). The crude product waspurified using preparative RPLC (CH₃CN:0.1M ammonium acetate (55:45)).The fractions of interest were partly concentrated and extracted withmethylene chloride. The organic layer was dried (Na₂SO₄) andconcentrated yielding 206 mg (87%) of a diastereomeric mixture.

[0478] LC-MS (m/z) 623 (M+1)⁺

[0479] (iv) 6,8-Dichloro-4-hydroxychroman-4-yl-C(O)-Aze-Pab×HOAc

[0480] The title compound was prepared according to the method describedin Example 19(vi) above from6,8-dichloro-4-hydroxychroman-4-yl-C(O)-Aze-Pab(Teoc) (150 mg; 0.24mmol; from step (iii) above) and Bu₄NF (0.10 g, 0.32 mmol). The crudematerial was purified using preparative RPLC (CH₃CN:0.1M ammoniumacetate (30:70)). Yield 45 mg (35%).

[0481]¹H-NMR (400 MHz; CD₃OD): δ 7.73 (m, 2H); 7.54 (m, 2H); 7.32 (m,2H); 7.23 (d, 1H); 4.65-4.40 (m, 4H); 4.30 (m, 2H); 4.17-3.97 (m, 1H);2.8-2.5 (m, 1H); 2.40 (m, 1H); 2.35-2.20 (m, 1H); 2.15 (m, 2H); 1.95 (s,3H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ 175.4;174.0; 174.3; 173.0; 168.1 LC-MS (m/z) 477 (M+1)⁺

Example 25 6-Fluoro-4-hydroxychroman-4-yl-C(O)-Aze-Pab×HOAc

[0482] (i) 6-Fluoro-4-hydroxychroman-4-yl-carboxylic Acid Methyl Ester

[0483] The sub-title compound was prepared according to the methoddescribed by Bigge et al (J. Med. Chem. (1993), 36, 1977ff) from6-fluorochromanone (2.53 g; 15.2 mmol), Me₃SiCN (1.66 g; 16.7 mmol), andZnI₂ (3 mg; cat.). Yield 2.51 g (73%).

[0484]¹H-NMR (400 MHz; CDCl₃): δ 6.93 (m, 1H); 6.82 (m, 2H); 4.34 (m,1H); 4.23 (dt, 1H); 3.81 (s, 3H); 2.47 (m, 1H); 2.10 (m, 1H)

[0485] (ii) 6-Fluoro-4-hydroxychroman-4-yl-carboxylic Acid

[0486] A solution of LiOH.H₂O (0.95 g; 22.6 mmol) in water (30 mL) wasadded to a solution of 6-fluoro-4-hydroxychroman-4-yl-carboxylic acid,methyl ester (2.47 g; 10.9 mmol; from step (i) above) in THF (10 mL).The reaction mixture was stirred at room temperature for 2 days, THF wasevaporated and the water phase was acidified with HCl (2M) and extractedwith ethyl acetate. The organic layer was dried (Na₂SO₄) and evaporated,yielding the sub-title compound. Yield: 1.41 g (61%).

[0487] LC-MS (m/z) 211 (M−1)⁻

[0488] (iii) 6-Fluoro-4-hydroxychroman-4-yl-C(O)-Aze-Pab(Z)

[0489] The sub-title compound was prepared according to the methoddescribed in Example 22(iii) above from6-fluoro-4-hydroxychroman-4-yl-carboxylic acid (250 mg; 1.18 mmol; fromstep (ii) above), HATU (500 mg; 1.32 mmol), H-Aze-Pab(Z)×HCl (570 mg;1.3 mmol; prepared according to the method described in InternationalPatent Application WO 97/02284) and 2,4,6-trimethylpyridine (0.70 g; 5.3mmol). The crude product was purified using preparative RPLC (CH₃CN:0.1Mammonium acetate; 55:45). The fractions of interest were partlyconcentrated and extracted with methylene chloride. The organic layerwas dried (Na₂SO₄) and concentrated yielding 290 mg (40%) of adiastereomeric mixture.

[0490] FAB-MS (m/z) 561 (M+1)⁺

[0491] (iv) 6-Fluoro-4-hydroxychroman-4-yl-C(O)-Aze-Pab×HOAc

[0492] HOAc (80 μL) and Pd/C (5%; 93 mg) were added to a solution of6-fluoro-4-hydroxychroman-4-yl-C(O)-Aze-Pab(Z) (140 mg; 0.25 mmol; fromstep (iii) above) in EtOH (10 mL), and the mixture was hydrogenated atambient temperature and pressure for 4 h. The mixture was filteredthrough Celite. The solution was concentrated and the crude material waspurified using preparative RPLC (CH₃CN:0.1M ammonium acetate (20:80)).Yield 72 mg (59%).

[0493]¹H-NMR (400 MHz; D₂O): δ 7.78 (dd, 1H); 7.73 (d, 1H); 7.55 (m,2H); 7.18-6.96 (m, 3H); 4.96 (dd, 1H); 4.58 (s, 1H); 4.50-4.35 (m, 2H);4.19 (m, 2H); 2.63 (m, 1H); 2.45 (m, 1H); 2.35-2.12 (m, 2H); 1.98 (s,3H) ¹³C-NMR (100 MHz; D₂O): (carbonyl and/or amidine carbons) δ 176.1;175.9; 174.7; 173.7; 167.6

Example 26 4-Hydroxy-6-methylchroman-4-yl-C(O)-Aze-Pab×HOAc

[0494] (i) 4-Hydroxy-6-methylchroman-4-yl-carboxylic Acid, Methyl Ester

[0495] The sub-title compound was prepared according to a methoddescribed by Bigge et al (J. Med. Chem. (1993) 36, 1977ff) from6-methylchromanone (3.11 g; 19.2 mmol), Me₃SiCN (2.1 g; 21.2 mmol), andZnI₂ (20 mg; cat.). Yield 2.80 g (62%).

[0496]¹H-NMR (300 MHz; CDCl₃): δ 7.01 (dd, 1H); 6.89 (d, 1H); 6.77 (d,1H); 4.37 (dt, 1H); 4.32-4.20 (m, 3H); 2.49 (m, 2H); 2.34 (s, 3H); 2.08(m, 1H); 1.24 (t, 3H)

[0497] (ii) 4Hydroxy-6-methylchroman-4-yl-carboxylic Acid

[0498] A solution of LiOH.H₂O (0.78 g; 18.6 mmol) in water (15 mL) wasadded to a solution of 4-hydroxy-6-methylchroman-4-yl-carboxylic acid,methyl ester (2.2 g; 9.3 mmol; from step (i) above) in THF (10 mL). Thereaction mixture was stirred at room temperature overnight, THF wasevaporated and the water phase was washed with ether. The resultantsolution was acidified with HCl (2M) and extracted with ether. Theorganic layer was dried (Na₂SO₄) and evaporated, yielding the sub-titlecompound. Yield: 1.21 mg (62%).

[0499]¹H-NMR (300 MHz; CD₃OD): δ 7.06 (d, 1H); 6.98 (d, 1H); 6.69 (d,1H); 4.32 (m, 1H); 4.17 (m, 1H); 2.50 (m, 1H); 2.21 (s, 3H); 2.03 (m,1H) LC-MS (m/z) 207 (M−1)⁻

[0500] (iii) 4Hydroxy-6-methylchroman-4-yl-C(O)-Aze-Pab(Z)

[0501] The sub-title compound was prepared according to a methoddescribed in Example 22(iii) above from4-hydroxy-6-methylchroman-4-yl-carboxylic acid (310 mg; 1.49 mmol; fromstep (ii) above), HATU (620 mg; 1.63 mmol), H-Aze-Pab(Z), (790 mg; 2.2mmol; prepared according to the method described in International PatentApplication WO 97/02284) and 2,4,6-trimethylpyridine (0.37 g; 3.0 mmol).The crude product was purified using preparative RPLC (CH₃CN:0.1Mammonium acetate (45:55)). The fractions of interest were partlyconcentrated and extracted with methylene chloride. The organic layerwas dried (Na₂SO₄) and concentrated yielding 675 mg (81%) of adiastereomeric mixture.

[0502] LC-MS (m/z) 557 (M+1)⁺

[0503] (iv) 4-Hydroxy-6-methylchroman-4-yl-C(O)-Aze-Pab×HOAc

[0504] HOAc (80 μL) and Pd/C (5%, 150 mg) were added to a solution of4-hydroxy-6-methylchroman-4-yl-C(O)-Aze-Pab(Z) (240 mg; 0.43mmol; fromstep (iii) above) dissolved in EtOH (10 mL), and the mixture washydrogenated at ambient temperature and pressure overnight. The mixturewas filtered through Celite. Freeze drying gave the title compound in ayield of 159 mg (76%).

[0505]¹H-NMR (500 MHz; CD₃OD): δ 7.72 (dd, 2H); 7.53 (dd, 2H); 7.08 (d,1H); 7.00 (d, 1H); 6.71 (d, 1H); 4.84 (m, partly hidden); 4.60 (m, 1H);4.46 (m, 1H); 4.29 (m, 2H); 4.14 (t, 1H); 2.40 (m, 2H); 2.26-2.10 (m,3H); 2.00 (m, 1H); 1.90 (s, 3H) ¹³C-NMR (100 MHz; CD₃OD): (carbonyland/or amidine carbons) δ 176.6; 174.5; 173.1; 168.1 LC-MS (m/z) 423(M+1)⁺

Example 27 8-Chloro-4-hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab×HOAc

[0506] (i) Ethyl 3-(2-chloro-4-methoxyphenoxy)propionate

[0507] Sodium (0.055 g; 2.4 mmol) and ethanol (1.5 mL) was added to amelt of 2-chloro-4-methoxyphenol (5.20 g; 32.8 mmol). When all thesodium was dissolved, ethyl acrylate (4.1 g; 41 mmol) was added and themixture was heated at 105° C. for 7 days. The mixture was then cooled toRT and partitioned between ether and water. The mixture was made acidicwith HCl (2M; aq.) and extracted with ether three times. The combinedorganic layer was washed with NaOH (2M; aq), dried (CaCl₂) andevaporated. The crude product (2.7 g) was purified using preparativeRPLC (CH₃CN:0.1M ammonium acetate (60:40)). Yield 1.90 g (22%).

[0508] (ii) 3-(2-Chloro-4-methoxyphenoxy)propionic Acid

[0509] A solution of LiOH.H₂O (0.67 g; 16 mmol) in water (20 mL) wasadded to a solution of ethyl 3-(2-chloro-4-methoxyphenoxy)propionate(1.90 g; 7.3 mmol; from step (i) above) in THF (10 mL). The reactionmixture was stirred at RT overnight, THF was evaporated and the waterphase was washed with ether. The resultant solution was acidified withHCl (2M) and extracted with ether. The organic layer was dried (Na₂SO₄)and evaporated yielding 0.90 g (54%) of the sub-title compound.

[0510] LC-MS (m/z) 229 (M−1)⁻

[0511] (iii) 8-Chloro-6-methoxychroman-4-one

[0512] Phosphorous pentachloride (1.3 g; 6.2 mmol) was added to asuspension of 3-(2-chloro-4-methoxyphenoxy)propionic acid (0.85 g; 3.7mmol; from step (ii) above) in benzene (10 mL). The resultant clearsolution was heated quickly to boiling and then cooled on an ice bath.Aluminium chloride (1.5 g; 11 mmol) was added in portions and, aftercomplete addition, ice water was added. Extraction with ether, washingof the organic layer with NaHCO₃/aq. and NaOH (2M; aq.), drying (Na₂SO₄)and concentration yielded 0.73 g (93%) of the sub-title compound.

[0513]¹H-NMR (300 MHz; CDCl₃): δ 7.27 (d, 1H); 7.19 (d, 2H); 4.59 (t,2H); 3.80 (s, 3H); 2.81 (t, 2H)

[0514] (iv) 8-Chloro-4-hydroxy-6-methoxychroman-4-yl-carboxylic Amide

[0515] The sub-title compound was obtained during an attempt to preparethe corresponding methyl ester according to the method described byBigge et al (J. Med. Chem. (1993) 36, 1977ff) from8-chloro-6-methoxychromanone (0.73 g; 3.4 mmol; from step (iii) above),Me₃SiCN (0.94 g; 7.6 mmol), and ZnI₂ (50 mg; cat.). The crude productconsisted of a minor amount of the corresponding methyl ester and amajor amount of the amide. The amide was purified by preparative RPLC(CH₃CN:0.1M ammonium acetate; 30:70 to 70:30). Yield 0.39 g (44%).

[0516] LC-MS (m/z) 256 (M−1)⁻

[0517] (v) 8-Chloro-4-hydroxy-6-methoxychroman-4-yl-carboxylic Acid

[0518] KOH (1.2 g; 21 mmol) and water (25 mL) were added to a solutionof 4-hydroxy-8-chloro-6-methoxychroman-4-yl-carboxylic amide (0.39 g;1.5 mmol; from step (iv) above) in i-PrOH (25 mL). The reaction mixturewas refluxed overnight, i-PrOH was evaporated and the water solution waswashed with ether. The reaction mixture was acidified with HCl (2M) andextracted with ethyl acetate. The organic layer was dried (Na₂SO₄) andevaporated. Yield: 0.38 mg (97%).

[0519] LC-MS (m/z) 257 (M−1)⁻

[0520] (vi) 8-Chloro-4-hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab(Teoc)

[0521] The sub-title compound was prepared according to the methoddescribed in Example 22(iii) above from4-hydroxy-8-chloro-6-methoxychroman-4-yl-carboxylic acid (260 mg; 1.00mmol; from step (v) above), HATU (420 mg; 1.1 mmol), H-Aze-Pab(Teoc)×HCl(490 mg; 1.1 mmol; see Example 19(iv) above), and2,4,6-trimethylpyridine (600 mg; 4.5 mmol). The crude product waspurified using preparative RPLC (CH₃CN:0.1M ammonium acetate (55:45)).The fractions of interest were partly concentrated and extracted withmethylene chloride. The organic layer was dried (Na₂S₄) andconcentrated, yielding 340 mg (55%) of a diastereomeric mixture.

[0522] LC-MS (m/z) 617 (M+1)⁺

[0523] (vii) 8-Chloro-4-hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab×HOAc

[0524] The title compound was prepared according to the method describedin Example 19(vi) above using4-hydroxy-8-chloro-6-methoxychroman-4-yl-C(O)-Aze-Pab(Teoc) (150 mg;0.24 mmol; from step (vi) above) and Bu₄NF (1.0M in THF; 0.32 mL). Thecrude material was purified using preparative RPLC (CH₃CN:0.1M ammoniumacetate (20:80)). Yield 113 mg (87%).

[0525]¹H-NMR (400 MHz; CD₃OD): δ 7.69 (d, 2H); 7.54 (d, 2H); 6.90 (d,1H); 6.85 (d, 1H); 4.57 (m, 3H); 4.48-4.30 (m, 4H); 4.17 (m, 2H); 4.00(m, 1H); 2.8-2.5 (m, 2H); 2.40 (m, 2H); 2.26 (m, 1H); 2.15 (m, 2H); 2.06(d, 1H) LC-MS (m/z) 473 (M−1)⁻

Example 28

[0526] 6-Chloro-4-hydroxy-8-methylchroman-4-yl-C(O)-Aze-Pab×HOAc

[0527] (i) Ethyl 3-(4-chloro-2-methylphenoxy)propionate

[0528] The sub-title compound was prepared according to the methoddescribed in Example 27(i) above from 4-chloro-2-methylphenol (4.99 g;35.0 mmol), sodium (0.055 g; 2.4 mmol), ethanol (1.5 mL) and ethylacrylate (4.1 g; 41 mmol). The crude product (1.98 g; 23%) was used forthe next step without further purification.

[0529] (ii) 3-(4-Chloro-2-methylphenoxy)propionic Acid

[0530] A solution of LiOH.H₂O (0.50 g; 12 mmol) in water (10 mL) wasadded to a solution of ethyl 3-(4-chloro-2-methylphenoxy)propionate(1.98 g; 8.15 mmol; from step (i) above) in THF (20 mL). The reactionmixture was stirred at room temperature overnight, THF was evaporatedand the water phase was washed with ether. The resultant solution wasacidified with HCl (2M), whereafter a solid material precipitated. Theproduct was filtered and air-dried yielding 0.62 g (35%) of thesub-title compound.

[0531] LC-MS (m/z) 213 (M−1)⁻

[0532] (iii) 6-Chloro-8-methylchroman-4-one

[0533] Phosphorous pentachloride (0.95 g, 4.6 mmol) was added to asuspension of 3-(4-chloro-2-methylphenoxy)propionic acid (0.59 g; 2.7mmol; from step (ii) above) in benzene (10 mL). The resultant clearsolution was heated quickly to boiling and then cooled on an ice bath.Aluminium chloride (1.0 g; 7.5 mmol) was added in portions and, aftercomplete addition, ice water was added. Extraction with ether, washingof the organic layer with NaHCO₃/aq. and NaOH (2M; aq.), drying (Na₂SO₄)and concentration yielded 0.27 g (50%) of the sub-title compound.

[0534]¹H-NMR (500 MHz; CDCl₃): δ 7.70 (d, 1H); 7.29 (d, 2H); 4.56 (t,2H); 2.79 (t, 2H); 2.22 (s, 3H)

[0535] (iv) 6-Chloro-4-hydroxy-8-methylchroman-4-yl-carboxylic Amide

[0536] The sub-title compound was prepared as described in Example27(iv) above, using the method described by Bigge et al (J. Med. Chem.(1993) 36, 1977ff) from 6-chloro-8-methylchromanone (0.27 g; 1.37 mmol;from step (iii) above), Me₃SiCN (0.29 g; 1.52 mmol), and ZnI₂ (46 mg;cat.). The crude product consisted of a minor amount of thecorresponding methyl ester and a major amount of the amide. The amidewas purified by preparative RPLC (CH₃CN:0.1M ammonium acetate; 30:70 to70:30). Yield: 0.17 g (50%).

[0537] LC-MS (m/z) 240 (M−1)⁻

[0538] (v) 6-Chloro-4-hydroxy-8-methylchroman-4-1-carboxylic Acid

[0539] KOH (1.25 g; 22.3 mmol) and water (20 mL) were added to asolution of 4-hydroxy-6-chloro-8-methylchroman-4-yl-carboxylic amide(0.17 g; 0.69 mmol; from step (iv) above) in i-PrOH (20 mL). Thereaction mixture was refluxed overnight, i-PrOH was evaporated and thewater solution was washed with ether. The reaction mixture was acidifiedwith HCl (2M) and extracted with ethyl acetate. The organic layer wasdried (Na₂SO₄) and evaporated. Yield: 0.13 g (78%).

[0540] (vi) 6-Chloro-4-hydroxy-8-methylchroman-4-yl-C(O)-Aze-Pab(Teoc)

[0541] A solution of 6-chloro-4-hydroxy-8-methylchroman-4-yl-carboxylicacid (130 mg; 0.54 mmol; from step (v) above) and HATU (220 mg; 0.59mmol) in DMF (5 mL) was stirred at 0° C. for 1.5 h, and a mixture ofH-Aze-Pab(Teoc)×HCl (270 mg; 0.59 mmol; see Example 19(iv) above) and2,4,6-trimethylpyridine (320 mL; 2.4 mmol) in DMF (3 mL) was added at 0°C. After stirring for 3 h at 0° C. the reaction mixture wasconcentrated, and the crude product was purified using preparative RPLC(CH₃CN:0.1M ammonium acetate (55:45)). The fractions of interest werepartly concentrated and extracted with methylene chloride. The organiclayer was dried (Na₂SO₄) and concentrated, yielding 79 mg (24%) of adiastereomeric mixture.

[0542] LC-MS (m/z) 601 (M−1)⁻

[0543] (vii) 6-Chloro-4-hydroxy-8-methylchroman-4-yl-C(O)-Aze-Pab×HOAc

[0544] Bu₄NF (1.0M in THF; 0.20 mL) was added to a solution of6-chloro-4-hydroxy-8-methylchroman-4-yl-C(O)-Aze-Pab(Teoc) (79 mg; 0.13mmol; from step (vi) above) in THF (5 mL) at 0° C. The solution wasstirred at 60° C. overnight, and was subsequently concentrated. Thecrude material was purified using preparative RPLC (CH₃CN:0.1M ammoniumacetate (20:80)). Yield 37 mg (54%).

[0545]¹H-NMR (400 MHz; CD₃OD): δ 7.72 (m, 2H); 7.54 (m, 2H); 7.15-6.98(m, 2H); 4.60 (m, 1H); 4.5-4.3 (m, 3H); 4.25-4.10 (m, 2H); 4.03 (m, 1H);2.80-2.45 (m, 1H); 2.37 (m, 1H); 2.26 (m, 1H); 2.14 (s, 3H); 2.05 (d,1H); 1.92 (s, 3H)

[0546] LC-MS (m/z) 473 (M−1)⁻

Example 29 (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-C(O)-i-Pr)

[0547] 2-Methylpropanoic anhydride (7.3 mg; 46 μmol) was added to anice-cold solution of (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(OH) (20 mg; 44 μmol;see Example 14 above) and Et₃N (4.9 mg; 49 μmol) in methylene chloride(1 mL), and the mixture was stirred at RT overnight. The mixture wasdiluted with a further amount of methylene chloride, washed 3 times withwater and once with brine, dried (Na₂SO₄) and concentrated. The crudeproduct was purified using preparative RPLC (CH₃CN:0.1M ammonium acetate(40:60)), and the fractions of interest were concentrated. Freeze dryingyielded 13 mg (56%) of the title compound.

[0548]¹H-NMR (300 MHz; CDCl₃): δ 7.90 (m, 1H); 7.65 (d, 2H); 7.29 (d,2H); 7.05 (d, 1H); 6.83 (dd, 1H); 6.67 (d, 1H); 5.13 (b, 2H); 4.93 (dd,1H); 4.48 (m, 3H); 3.84 (m, 1H); 3.76 (s, 3H); 3.03 (m, 1H); 2.85-2.70(m, 2H); 2.5-2.7 (m, 2H); 2.25 (m, 1H); 2.00-1.93 (m, 4H); 1.29 (d, 6H)¹³C-NMR (75 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ 177.7;174.3; 170.3 LC-MS (m/z) 523 (M+1)⁺

Example 30 (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-C(O)-Et)

[0549] Propanoic anhydride (9.5 mg; 73 μmol) was added to an ice-coldsolution of (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(OH) (30 mg; 66 μmol;see Example 14 above) and Et₃N (7.4 mg; 73 μmol) in methylene chloride(1 mL). The mixture was stirred at RT overnight. The crude product waspurified using preparative RPLC (CH₃CN:0.1M ammonium acetate; 30:70 to40:60) and the fractions of interest were concentrated. Freeze dryingyielded 19 mg (56%) of the title compound.

[0550]¹H-NMR (400 MHz; CDCl₃): δ 7.93 (t, 1H); 7.67 (d, 2H); 7.32 (d,2H); 7.06 (d, 1H); 6.83 (dd, 1H); 6.68 (d, 1H); 5.12 (b, 2H); 4.93 (dd,1H); 4.50 (m, 2H); 3.84 (m, 1H); 3.76 (s, 3H); 3.03 (m, 1H); 2.67-2.50(m, 2H); 2.5-2.7 (m, 4H); 2.26 (m, 1H); 1.92 (m, 4H); 1.26 (t, 3H)¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ 178.8;173.1; 171.4 LC-MS (m/z) 509 (M+1)⁺

Example 31 (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-C(O)-Ch)

[0551] Cyclohexanecarboxylic chloride (7.3 mg; 46 μmol) was added to anice-cold solution of (8)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(OH) (30 mg; 66 μmol;see Example 14 above) and Et₃N (7 mg; 73 μmol) in methylene chloride (1mL). The mixture was stirred at RT overnight. The mixture was dilutedwith a further amount of methylene chloride, and the mixture was washed3 times with water and once with brine, dried (Na₂SO₄) and concentrated.The crude product was purified using preparative RPLC (CH₃CN:0.1Mammonium acetate (40:60)) and the fractions of interest wereconcentrated. Freeze drying yielded 18 mg (50%) of the title compound.

[0552]¹-NMR (400 MHz; CDCl₃): δ 7.91 (t, 1H); 7.67 (d, 2H); 7.30 (d,2H); 7.06 (d, 1H); 6.83 (m, 1H); 6.67 (d, 1H); 5.09 (b, 2H); 4.93 (dd,1H); 4.50 (m, 3H); 3.83 (m, 1H); 3.76 (s, 3H); 3.02 (q, 1H); 2.68-2.45(m, 3H); 2.26 (m, 1H); 2.1-1.9 (m, 6H); 1.83 (m, 2H); 1.70 (m, 1H); 1.59(m, 2H); 1.32 (m, 3H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidinecarbons) δ 178.7; 174.2; 171.4 LC-MS (m/z) 563 (M+1)⁺

Example 32 (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-allyl)

[0553] (i) (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Teoc)

[0554] The sub-title compound was prepared according to the methoddescribed in Example 22(iii) above from1-hydroxy-7-methoxytetralin-1-yl-carboxylic acid (0.44 g; 2.0 mmol; seeExample 1(ii) above), HATU (0.80 g; 2.1 mmol), H-Aze-Pab(Teoc)×HCl,(1.17 g; 2.6 mmol; see Example 19(iv) above) and 2,4,6-trimethylpyridine(1.2 g; 10 mmol). The crude product (1.73 g) was purified usingpreparative RPLC (CH₃CN:0.1M ammonium acetate; 55:45 to 45:55). Thefractions of interest were partly concentrated and extracted withmethylene chloride. The organic layer was dried (Na₂SO₄) andconcentrated yielding 0.32 g (28%) of a diastereomeric mixture.Preparative RPLC (CH₃CN:0.1M ammonium acetate (46:54)) yielded twodiastereomers: Compound 32A (faster moving diastereomer; 0.16 g; 28%)and Compound 32B (slower moving diastereomer; 0.16 g; 28%).

[0555] Compound 32A:

[0556]¹H-NMR (400 MHz; CDCl₃): δ 7.96 (t, 1H); 7.86 (dd, 2H); 7.36 (dd,2H); 7.07 (d, 1H); 6.87 (dd, 1H); 6.68 (d, 1H); 4.95 (dd, 1H); 4.54 (m,3H); 4.26 (m, 2H); 3.84 (m, 1H); 3.78 (s, 3H); 3.04 (q, 1H); 2.83 (d,1H); 2.63 (m, 2H); 2.28 (m, 1H); 2.02-1.85 (m, 4H); 1.15 (dt, 2H); 0.08(s, 9H) LC-MS (m/z) 581 (M+1)⁺

[0557] (ii) (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Teoc)(O-allyl)

[0558] O-Allylhydroxylamine×HCl (57 mg; 0.52 mmol) was added to asolution of (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Teoc) (50 mg; 86 μmol;Compound 32A from step (i) above) in THF (3 mL), and the mixture wasstirred at 60° C. overnight. The solution was concentrated, and thecrude product was purified using preparative RPLC (CH₃CN:0.1M ammoniumacetate; 55:45 to 60:40). The fractions of interest were concentrated,and the remaining mixture was extracted with methylene chloride. Theorganic layer was washed with brine, dried (Na₂SO₄), and concentratedyielding 28 mg (51%) of the sub-title compound.

[0559]¹H-NMR (400 MHz; CDCl₃): δ 7.81 (t, 1H); 7.59 (s, 1H); 7.48 (d,2H); 7.30 (d, 2H); 7.06 (d, 1H); 6.83 (dd, 1H); 6.69 (d, 1H); 6.04 (m,1H); 5.35 (m, 1H); 5.27 (d, 1H); 4.92 (dd, 1H); 4.66 (dd, 1H); 4.50 (m,1H); 4.16 (m, 2H); 3.81 (m, 1H); 3.78 (s, 3H); 2.97 (q, 1H); 2.82 (d,1H); 2.60 (m, 2H); 2.26 (m, 1H); 2.05-1.85 (m, 4H); 0.98 (m, 2H); 0.03(s, 9H)

[0560] (iii) (S)- or(R)-l1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-allyl)

[0561] The title compound was prepared according to the method describedin Example 19(vi) from (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Teoc)(O-allyl) (28 mg;44 μmol; from step (ii) above) in CH₃CN (2 mL) and Bu₄NF (1M in THF; 0.1mL; 0.1 mmol). The crude product (21.3 mg) was purified using flashchromatography (Si gel; ethyl acetate). yielding 10 mg (46%).

[0562]¹H-NMR (400 MHz; CDCl₃): δ 7.88 (t, 1H); 7.62 (d, 2H); 7.30 (d,2H); 7.06 d, 1H); 6.83 (dd, 1H); 6.68 (d, 1H); 6.09 (m, 1H); 5.35 (m,1H); 5.23 (m, 1H); 4.93 (dd, 1H); 4.84 (s, 3H); 4.68 (m, 1H); 4.50 (m,2H); 3.82 (m, 1H); 3.77 (s, 3H); 3.01 (m, 1H); 2.82 (d, 1H); 2.62 (m,2H); 2.26 (m, 1H); 2.0-1.8 (m, 4H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyland/or amidine carbons) δ 178.8; 171.2; 159.6 LC-MS: (m/z) 493 (M+1)⁺

Example 33 (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-Bzl)

[0563] (i) (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Teoc)(O-Bzl)

[0564] O-Benzylhydroxylamine×HCl (82 mg; 0.52 mmol) was added to asolution of (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Teoc) (50 mg; 86 μmol;see Example 32(i) above) in THF (3 mL), and the mixture was stirred at60° C. overnight. The solution was concentrated, and the crude productwas purified using preparative RPLC (CH₃CN:0.1M ammonium acetate; 60:40to 70:30). The fractions of interest were concentrated, and theremaining mixture was extracted with methylene chloride. The organiclayer was washed with brine, dried (Na₂SO₄), and concentrated yielding41 mg (70%) of the sub-title compound.

[0565]¹H-NMR (400 MHz; CDCl₃): δ 7.81 (t, 1H); 7.60 (s, 1H); 7.47 (d,2H); 7.40 (m, 5H); 7.30 (d, 2H); 7.06 (d, 1H); 6.83 (dd, 1H); 6.69 (d,1H); 5.18 (s, 2H); 4.92 (dd, 1H); 4.51 (m, 2H); 4.15 (m, 2H); 3.81 (m,1H); 3.77 (s, 3H); 2.81 (d, 1H); 2.60 (m, 2H); 2.25 (m, 1H); 2.1-1.8 (m,4H); 0.96 (m, 2H); 0.02 (s, 9H) LC-MS (m/z) 687 (M+1)⁺

[0566] (ii) (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-Bzl)

[0567] The title compound was prepared according to the method describedin Example 19(vi) above from (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Teoc)(O-Bzl) (28 mg;44 μmol; from step (i) above) and Bu₄NF (1M in THF; 0.1 mL; 0.1 mmol).The crude product (21 mg) was purified using flash chromatography (Sigel; ethyl acetate). Yield: 10 mg (35%).

[0568]¹H-NMR (400 MHz; CDCl₃): δ 7.88 (t, 1H); 7.61 (d, 2H); 7.45 (d,2H); 7.40-7.35 (m, 5H); 7.06 (d, 1H); 6.83 (dd, 1H); 6.68 (d, 1H); 5.15(s, 2H); 4.92 (dd, 1H); 4.85 (b, 2H); 4.50 (b+m, 3H); 3.83 (m, 1H); 3.77(s, 3H); 3.02 (m, 1H); 2.82 (d, 1H); 2.62 (m, 2H); 2.26 (m, 1H); 2.0-1.8(m, 4H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ178.8; 171.3; 159.6 LC-MS (m/z) 543 (M+1)⁺

Example 34 (S)- or (R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(CO-O-methallyl)

[0569] (i) p-Nitrophenyl-methallyl Carbonate

[0570] Pyridine (1.21 g; 15 mmol) was added to an ice-cold solution ofmethallyl alcohol (1.0 g; 14 mmol) and p-nitrophenyl chloroformate (3.07g; 15 mmol) in methylene chloride (40 mL), and the resultant mixture wasstirred at RT for 1 hour, whereafter the solution was washed with KHSO₄(3×) and brine, dried (Na₂SO₄), and concentrated to yield 2.9 g (88%) ofthe sub-title compound.

[0571]¹H-NMR (400 MHz; CDCl₃): δ 8.29 (d, 2H); 7.40 (d, 2H); 5.12 (s,1H); 5.06 (s, 1H); 4.70 (s, 2H); 1.85 (s, 3H)

[0572] (ii) (S)- or (R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(CO-O-methallyl)

[0573] NaOH (aq.; 2M; 0.35 ml; 0.7 mmol) was added to an ice-coldsolution of (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab×HOAc (32 mg; 64 μmol;see Example 3 above) in THF (3 mL), whereafter p-nitrophenyl-methallylcarbonate (17 mg; 71 μmol; from step (i) above) was added and thesolution was stirred at RT for 1 hour. The crude product was purifiedusing preparative RPLC (CH₃CN:0.1M ammonium acetate (40:60)). Thefractions of interest were concentrated and the water solution wasextracted with methylene chloride. The organic layer was washed withbrine, dried (Na₂SO₄), and concentrated. The product was dissolved inCH₃CN/water and freeze dried to yield 23 mg (67%) of the title compound.

[0574]¹H-NMR (400 MHz; CDCl₃): δ 7.97 (t, 1H); 7.83 (d, 2H); 7.33 (d,2H); 7.06 (d, 1H); 6.83 (dd, 1H); 6.67 (d, 1H); 5.06 (s, 2H); 4.93 (m,2H); 4.60 (s, 2H); 4.51 (m, 2H); 3.84 (m, 1H); 3.76 (s, 3H); 3.05 (m,1H); 2.82 (d, 1H); 2.60 (m, 2H); 2.27 (m, 1H); 2.0-1.85 (m, 4H); 1.83(s, 3H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ178.8; 171.4; 159.6 LC-MS (m/z) 535 (M+1)⁺

Example 35 1-Hydroxy-7-aminotetralin-1-yl-C(O)-Aze-Pab(OH)

[0575] (i) 1-Hydroxy-7-nitrotetralin-1yl-C(O)-Aze-Pab(Teoc)

[0576] The sub-title compound was prepared according to the methoddescribed in Example 22(iii) above from1-hydroxy-7-nitrotetralin-1-yl-carboxylic acid (200 mg; 0.84 mmol; seeExample 7(ii) above), HATU (353 mg; 0.93 mmol), H-Aze-Pab(Teoc) (417 mg,0.93 mmol; see Example 19(iv) above) and 2,4,6-trimethylpyridine (409mg; 3.37 mmol). The crude product was purified using preparative RPLC(CH₃CN:0.1M ammonium acetate (50:50)). The fractions of interest wereconcentrated and freeze dried to yield 226 mg (45%) of the sub-titlecompound.

[0577]¹H-NMR (400 MHz; CDCl₃): δ 8.04 (m, 2H); 7.84 (d, 2H); 7.77 (d,1H); 7.29 (m, 2H); 4.93 (m, 1H); 4.65-4.50 (m, 1H); 4.40 (dd, 1H); 3.96(m, 1H); 3.82 (m, 5H); 3.15 (m, 1H); 2.95 (m, 1H); 2.75 (m, 1H); 2.52(m, 1H); 2.44-2.25 (m, 1H); 2.1-1.9 (m, 5H); 0.05 (s, 9H) LC-MS (m/z)596 (M+1)⁺

[0578] (ii) 1-Hydroxy-7-aminotetralin-1-yl-C(O)-Aze-Pab(Teoc)

[0579] A mixture of 1-hydroxy-7-nitrotetralin-1-yl-C(O)-Aze-Pab(Teoc)(48 mg; 81 μmol; from step (i) above), acetic acid (5 mg; 81 μmol), andPd/C (5%; 24 mg) was hydrogenated at ambient temperature and pressurefor 3 h. The resultant mixture was filtered through Celite, andconcentrated to yield 37 mg (85%) of the title compound.

[0580]¹H-NMR (400 MHz; CDCl₃): δ 7.86 (dd, 2H); 7.42 (d, 1H); 7.33 (d,1H); 6.89 (dd, 1H); 6.58 (dd, 1H); 6.47 (b, 0.5H); 6.23 (b, 0.5H); 4.91(m, 1H); 4.68-4.52 (m, 1H); 4.5-4.4 (m, 1H); 4.23 (m, 2H); 3.85 (m, 1H);3.69 (m, 1H); 3.2-3.0 (m, 1H); 2.74 (d, 1H); 2.65-2.45 (m, 2H); 2.4-2.2(m, 1H); 2.0-1.8 (m, 5H); 0.05 (s, 9H) LC-MS (m/z) 566 (M+1)⁺

[0581] (iii) 1-Hydroxy-7-aminotetralin-1-yl-C(O)-Aze-Pab(OH)

[0582] A mixture of hydroxylamine×HCl (29 mg; 41 mmol) and TEA (140 mg;1.38 mmol) in THF (10 mL) was sonicated at 40° C. for 1 h. A solution of1-hydroxy-7-aminotetralin-1-yl-C(O)-Aze-Pab(Teoc) (140 mg; 1.38 mmol;from step (ii) above) in THF (5 mL) was added, and the mixture wasstirred at 40° C. for 3 days. The resultant mixture was concentrated andthe crude product was purified using preparative RPLC (CH₃CN:0.1Mammonium acetate (30:70)). Concentration and freeze drying of thesolution yielded 20 mg (65%) of the title product.

[0583]¹H-NMR (400 MHz; CDCl₃): δ 8.26 (b, 0.5H); 8.03 (b, 0.5H); 7.57(dd, 2H); 7.39 (d, 1H); 7.30 (d, 1H); 6.91 (dd, 1H); 6.65-6.55 (m, 1H);4.98 (m, 3H); 4.65-4.30 (m+b, 4H); 3.88 (m, 0.5H); 3.69 (m, 0.5H); 3.14(m, 1H); 2.77 (d, 1H); 2.65-2.50 (m, 2H); 2.45-2.25 (m, 1H(; 2.10 (s,2H); 2.00-1.85 (m, 4H) ¹³C-NMR (100 MHz; CD₃OD): (carbonyl and/oramidine carbons) δ 178.2; 172.9; 155.2 LC-MS (m/z) 438 (M+1)⁺

Example 36 (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-Val)

[0584] (i) (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-Val(Boc))

[0585] EDC×HCl (16 mg; 83 μmol) was added to an ice-cold solution of(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(OH) (30 mg;66,μmol; see Example 14 above), Boc-Val-OH (18 mg; 83 μmol) and DMAP (24mg, 0.20 mmol) in DMF (3 mL), and the solution was stirred overnight.The resultant mixture was poured into water (200 mL), and the mixturewas extracted 3 times with EtOAc. The combined organic phases werewashed with dilute citric acid solution and brine, dried (Na₂SO₄), andconcentrated. The crude product (41 mg) was purified using preparativeRPLC (CH₃CN:0.1M ammonium acetate (40:60)). Yield 13 mg (30%).

[0586]¹H-NMR (500 MHz; CDCl₃): δ 7.94 (bt, 1H); 7.68 (d, 2H); 7.33 (d,2H); 7.08 (d, 1H); 6.85 (dd, 1H); 6.69 (d, 1H); 5.30 (b, 2H); 5.18 (bd,1H); 4.95 (m, 1H); 4.60-4.55 (m, 3H); 4.48 (dd, 1H); 4.32 (m, 1H); 3.86(m, 1H); 3.79 (s, 3H); 3.05 (m, 1H); 2.83 (m, 1H); 2.7-2.55 (m, 2H);2.28 (m, 1H); 2.22 (m, 1H); 2.05-1.85 (m, 5H); 1.48 (s, 9H); 1.08 (d,3H); 1.04 (d, 3H) LC-MS (m/z) 652 (M+1)⁺

[0587] (ii) (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-Val)

[0588] An ice-cold solution of1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-Val(Boc)) (12 mg; 18μmol; from step (i) above) in EtOAc saturated with HCl (5 mL) wasstirred for 80 minutes, whereafter the solution was concentrated,dissolved in water and freeze-dried overnight to yield 11 mg (96 %) ofthe title compound.

[0589]¹H-NMR (400 MHz; D₂O): δ 7.66 (d, 1H, minor); 7.59 (d, 2H, major);7.45-7.35 (m, 2H); 7.2-7.1 (m, 1H); 6.95-6.85 (m, 1H); 6.75-6.65 (m,1H); 5.25 (m, 1H, minor); 4.89 (m, 1H, major); 4.6-4.3 (m, 3H); 4.21 (m,1H); 4.14 (m, 1H, major); 3.87 (m, 1H, major); 3.81 (s, 3H, minor); 3.63(s, 3H, major); 2.8-1.7 (m, 9H); 1.11 (d, 6H) ¹³C-NMR (100 MHz; D₂O):(carbonyl and/or amidine carbons) δ 178.3; 173.8; 169.1; 161.4 LC-MS(m/z) 552 (M+1)⁺

Example 37 (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-N(Me)-Bzl-4-C(NH₂)NH×HOAc

[0590] (i) Methyl-4-cyanobenzylideneimine

[0591] A solution of p-cyanobenzaldehyde (13.1 g; 0.1 mol), methylamine(3.1 g; 0.1 mol) and p-TsOH (50 mg; cat.) in toluene (150 mL) wasstirred at RT overnight, whereafter it was washed with NaHCO₃/(aq. (2×)and brine, dried (Na₂SO₄) and concentrated. Yield 14.4 g (100%).

[0592]¹H-NMR (300 MHz; CDCl₃): δ 8.2 (s, 1H); 7.78 (d, 2H); 7.68 (d,2H); 3.54 (s, 3H)

[0593] (ii) Methyl-4-cyanobenzylamine

[0594] NaBH₄ (4.54 g; 0.42 mol) was added in portions to an ice-coldsolution of methyl-4-cyanobenzylideneimine (14.4 g; 0.1 mol; from step(i) above) in EtOH. The solution was stirred at RT overnight and theresultant solution was quenched with HCl (2M; aq.), washed with ether(2×), made alkaline with NaOH (2M; aq.) to pH 10, and extracted withEtOAc (3×). The organic solution was washed with water and brine, dried(Na₂SO₄), and concentrated. Yield 11.4 g (78%).

[0595]¹H-NMR (300 MHz; CDCl₃): δ 7.92 (d, 2H); 7.76 (d, 2H); 4.82 (s+b,5H); 4.40 (s, 2H)

[0596] (iii) Boc-Aze-N(Me)-Bzl-4-CN

[0597] EDC×HCl (14.5 g; 76 mmol) was added in portions to an ice-coldsolution of methyl-4-cyanobenzylamine (11.4 g; 78 mmol), Boc-Aze(OH)(15.4 g; 78 mmol) and DMAP (10.5 g; 82 mmol) in CH₃CN (500 mL),whereafter the mixture was stirred at RT overnight. The resultantmixture was partitioned between EtOAc and water, the aqueous phase wasextracted with EtOAc (3×100 mL), and the combined organic layer waswashed with NaHSO₄ (2×), water (2×) and brine (1×), dried (Na₂SO₄) andconcentrated. The yield of the crude product was 23.2 g (90%). A smallamount (6.17 g; 18.7 mmol) was purified using flash chromatography (Sigel; EtOAc). Yield 4.0 g (65%).

[0598]¹H-NMR (400 MHz; CDCl₃) (complex due to rotamers): δ 7.66 (d, 2H,minor); 7.60 (d, 2H, major); 7.38 (d, 2H, major); 7.31 (d, 2H, minor);5.01 (dd, 1H); 4.9-4.7 (b, 1H); 4.6-4.45 (b, 1H); 4.07 (m, 1H); 3.90 (m,1H); 3.00 (s, 3H, minor); 2.96 (s, 3H, major); 2.46 (m, 1H); 1.43 (s,3H)

[0599] (iv) Aze-N(Me)-Bzl-4-CN×HCl

[0600] A solution of Boc-Aze-N(Me)-Bzl-4-CN (4.0 g; 12 mmol; from step(iii) above) in EtOAc (saturated with HCl; 50 mL) was stirred for 15min, whereafter the solution was concentrated. Yield 3.1 g (quant.)

[0601]¹H-NMR (400 MHz; D₂O): δ 7.80 (m,2H); 7.45 (m, 2H); 5.6-5.45 (m,1H); 4.72 (s, 2H); 4.3-4.1 (m, 1H); 4.08-3.95 (m, 1H); 2.94 (s, 3H);2.8-2.55 (m, 1H)

[0602] (v) (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-N(Me)-Bzl4-CN

[0603] A solution of Aze-N(Me)-Bzl-4-CN×HCl (0.56 g; 2.1 mmol; from step(iv) above) and 2,4,6-trimethylpyridine (0.51 g, 4.2 mmol) in DMF (3 mL)was added to an ice-cold solution of1-hydroxy-7-methoxytetralin-1-yl-carboxylic acid (0.44 g; 2.0 mmol; seeExample 1(ii) above) and HATU (0.80 g; 2.1 mmol) in DMF (3 mL), and themixture was stirred at RT overnight. The resultant mixture was pouredonto water (0.5 L) and extracted with EtOAc (3×). The organic solutionwas washed with brine, dried (Na₂SO₄), and evaporated. The crude product(1.06 g) was purified using prep. RPLC (CH₃CN:0.1M ammonium acetate(32.5:67.5)), yielding two diastereoisomers, a faster diastereoisomer(Compound 37A; yield 215 mg (50%)), and a slower diastereoisomer(Compound 37B; yield 205 mg (48%)).

[0604] Compound 37A

[0605]¹H-NMR (400 MHz; CDCl₃) (complex due to rotamerism): δ 7.74 (d,2H, minor); 7.66 (d, 2H, major); 7.42 (d, 2H, minor); 7.39 (d, 2H,major); 7.07 (m, 1H); 6.87-6.81 (m, 1H); 6.80 (d, 2H, major); 6.75 (d,2H, minor); 5.22 (dd, 1H, major); 5.02 (dd, 2H, minor); 4.71 (dd, 2H);4.60 (m, 1H); 3.98 (m, 1H); 3.81 (s, 3H, major); 3.78 (s, 3H, minor);3.05-2.05 (m, 4H; thereof 3.05, s, 3H, minor and 3.01, s, 3H, major);2.92-2.82 (m, 1H); 2.67 (m, 1H); 2.40 (m, 1H, minor); 2.25-2.07 (m, 3H);1.98 (m, 2H)

[0606] (vi) (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-N(Me)-Bzl-4-C(NH₂)NOH

[0607] A solution of (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-N(Me)-Bzl-4-CN (0.18 g;0.42 mmol; Compound 37A from step (v) above), hydroxylamine×HCl (88 mg;1.3 mmol) and TEA (0.18 mL; 1.3 mmol) in ethanol (abs., 3 mL) wasstirred at RT for 36 h, whereafter the crude product was purified usingflash chromatography (methylene chloride:methanol (90:10)). The combinedfractions of interest were concentrated. Yield 0.18 g (91%).

[0608]¹H-NMR (400 MHz; CDCl₃) (complex due to rotamerism): δ 7.67 (d,2H, minor); 7.60 (d, 2H, major); 7.28 (m, 2H, partly obscured by CHCl₃);7.04 (dd, 1H); 6.85-6.72 (m, 2H); 5.65 (b, 2H, major); 5.33 (b, 2H,minor); 5.20 (dd, 1H, major); 5.06 (dd, 1H, minor); 4.75-4.45 (m, 3H);3.95 (m, 1H); 3.78 (s, 3H, major); 3.75 (s, 3H, minor); 3.00 (s, 3H,minor); 2.94 (d, 3H, major); 2.9-2.75 (m, 1H); 2.65 (m, 1H); 2.40 (m,2H, major); 2.10 (m, 3H); 1.95 (m, 2H) LC-MS (m/z) 467 (M+1)⁺

[0609] (vii) (S)- or(R)-1-Hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-N(Me)-Bzl-4-C(NH₂)NH×HOAc

[0610] A mixture of (S)- or(R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-N(Me)-Bzl-4-C(NH₂)NOH (60mg; 0.13 mmol; from step (vi) above), HOAc (15 mg; 0.26 mmol) and Pd/C(10%; 27 mg) in ethanol was chromatographed for 2 days, whereafter themixture was filtered through Celite. The resultant solution wasconcentrated, and the crude product was purified using preparative RPLC(CH₃CN:0.1M ammonium acetate; 10:90 to 20:80). The fractions of interestwere partially concentrated and freeze dried overnight.

[0611] Yield 18 mg (27%).

[0612]¹H-NMR (400 MHz; D₂O) (complex due to rotamerism): δ 7.83-7.73 (m,1H); 7.68 (d, 1H); 7.50 (t, 1H); 7.43 (d, 1H); 7.17 (d, 1H); 6.93 (m,1H); 6.82 (d, 1H); 5.40 (dd, 1H); 4.85 (d, 1H); 4.70 (m, 1H); 4.58 (d,1H); 4.4-4.0 (m, 2H); 3.70 (s, 3H); 3.10 (s, 3H); 2.9-2.6 (m, 3H);2.25-2.05 (m, 4H); 2.0-1.7 (m, 4H) ¹³C-NMR (100 MHz; D₂O): (carbonyland/or amidine carbons) (complex due to rotamerism) δ 178.6; 177.6;173.8; 173.3; 173.1; 167.5; 158.5; 158.4; 158.2 LC-MS (m/z) 451 (M+1)⁺

Example 38 9-Hydroxyfluoren-9-yl-C(O)-Aze-Pab×HOAc

[0613] (i) 9-Hydroxyfluoren-9-yl-C(O)-Aze-Pab(Z)

[0614] The sub-title compound was prepared according to the methoddescribed in Example 3(i) above from 9-hydroxyfluoren-9-yl-carboxylicacid (230 mg; 1.0 mmol), TBTU (350 mg; 1.1 mmol), H-Aze-Pab(Z)×HCl (500mg; 1.25 mmol; prepared according to the method described inInternational Patent Application WO 97/02284) and DIPEA (0.52 g; 4.0mmol). The crude product was purified using preparative RPLC (CH₃CN: 0.1M ammonium acetate; 50:50). The fractions of interest were partlyconcentrated and were extracted with EtOAc (3×). The organic layer wasdried (Na₂SO₄) and concentrated yielding 266 mg (46%) of the sub-titlecompound.

[0615]¹H-NMR (400 MHz; CDCl₃): δ 7.92 (d, 3H); 7.66 (dd, 2H); 7.5-7.2(m, 11H); 5.25 (s, 3H); 4.85 (dd, 1H); 4.52 (m, 2H); 2.83 (t, 2H); 2.33(m, 1H); 2.12 (m, 1H) LC-MS (m/z) 575 (M+1)⁺

[0616] (ii) 9-Hydroxyfluoren-9-yl-C(O)-Aze-Pab×HOAc

[0617] Pd/C (5%; 100 mg) and HOAc (9 μL) were added to a mixture of9-hydroxyfluoren-9-yl-C(O)-Aze-Pab(Z) (70 mg; 0.12 mmol; from step (i)above) in EtOH (10 mL). The mixture was hydrogenated at ambienttemperature and pressure for 6 h. The mixture was filtered throughCelite, concentrated and dissolved in water, whereafter the aqueoussolution was freeze dried. Yield 53 mg (88%).

[0618]¹H-NMR (400 MHz; D₂O): δ 7.9-7.65 (m, 4H); 7.60-7.35 (m, 8H); 4.51(s, 1H); 4.05 (m, 2H); 3.25 (t, 1H); 2.49 (m, 0.5H, rotamer); 2.28 (m,0.5H, rotamer); 1.98-1.84 (m, 7H; within this: 1.95, s, 3H) ¹³C-NMR (100MHz; D₂O): (carbonyl and/or amidine carbons) δ 173.4; 173.0; 172.6;167.0 FAB-MS (m/z) 441 (M+1)⁺

Example 39

[0619] The title compounds of Examples 1 to 12, 19 to 28, 37 and 38(which are all compounds of formula I) were tested in Test A above andwere all found to exhibit an IC₅₀TT value of less than 0.3 μM.

Example 40

[0620] The title compounds of Examples 13 to 18 and 29 to 36 (which areall compounds of formula Ia) were tested in Test A above and were allfound to exhibit an IC₅₀TT value of more than 1 μM.

Example 41

[0621] The title compounds of Examples 13 to 18 and 29 to 36 (which areall compounds of formula Ia) were tested in Test E above and were allfound to exhibit oral and/or parenteral bioavailability in the rat asthe corresponding active inhibitor of formula I. Ac = acyl AcOH = aceticacid Aze = azetidine-2-carboxylate AzeOH = azetidine-2-carboxylic acidDCC = dicyclohexylcarbodiimide DIPEA = diisopropylethylamine DMAP =N,N-dimethyl amino pyridine DMF = dimethylformamide DMSO =dimethylsulphoxide EDC = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride Et = ethyl Et₂O = diethyl ether EtOAc = ethyl acetate EtOH= ethanol h = hours HATU =O-(azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate HBTU =[N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium hexafluorophosphate]HCl(g) = hydrogen chloride gas HOAc = acetic acid LC = liquidchromatography Me = methyl MeOH = methanol Pab-H =para-amidinobenzylamino H-Pab-H = para-amidinobenzylamine QF =tetrabutylammonium fluoride (Bu₄NF) RPLC = preparative reverse phasehigh performance liquid chromatography RT = room temperature TBTU =[N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate]TEA = triethylamine Teoc = 2-(trimethylsilyl)ethoxycarbonyl THF =tetrahydrofuran TLC = thin layer chromatography Val = L-valine Z =benzyloxycarbonyl

[0622] Prefixes n, s, i and t have their usual meanings: normal, iso,secondary and tertiary.

1. A compound of formula I,

wherein R¹ represents H, C₁₋₄ alkyl (optionally substituted by one ormore substituents selected from cyano, halo, OH, C(O)OR^(1a) orC(O)N(R^(1b))R^(1c)) or OR^(1d); R^(1d) represents H, C(O)R¹¹,SiR¹²R¹³R¹⁴ or C₁₋₆ alkyl, which latter group is optionally substitutedor terminated by one or more substituent selected from OR¹⁵ or(CH₂)_(q)R¹⁶; R¹², R¹³ and R¹⁴ independently represent H, phenyl or C₁₋₆alkyl; R¹⁶ represents C₁₋₄ alkyl, phenyl, OH, C(O)OR¹⁷ or C(O)N(H)R¹⁸;R¹⁸ represents H, C₁₋₄ alkyl or CH₂C(O)OR¹⁹; R¹⁵ and R¹⁷ independentlyrepresent H, C₁₋₆ alkyl or C₁₋₃ alkylphenyl; R^(1a), R^(1b), R^(1c), R¹¹and R¹⁹ independently represent H or C₁₋₄ alkyl; and q represents 0, 1or 2; R_(x) represents a structural fragment of formula IIa, IIb or IIc,

wherein the dotted lines independently represent optional double bonds;A and B independently represent O or S, CH or CH₂ (as appropriate), or Nor N(R²¹) (as appropriate); D represents —CH₂—, O, S, N(R²²), —(CH₂)₂—,—CH═CH—, —CH₂N(R²²)—, —N(R²²)CH₂—, —CH═N—, —N═CH—, —CH₂O—, —OCH₂—,—CH₂S— or —SCH₂—; X₁ represents C₂₋₄ alkylene; C₂₋₃ alkylene interruptedby Z; —C(O)-Z-A¹; -Z-C(O)-A¹-; —CH₂—C(O)-A¹; -Z-C(O)-Z-A²-;—CH₂-Z-C(O)-A²-; -Z-CH₂—C(O)-A²-; -Z-CH₂-S(O)_(m)-A²-;—CH₂-Z-S(O)_(m)-A²-; —C(O)-A³; -Z-A³-; or -A³-Z-; X₂ represents C₂₋₃alkylene, —C(O)-A⁴- or -A⁴-C(O)—; X₃ represents CH or N; X₄ represents asingle bond, O, S, C(O), N(R²³), —CH(R²³)—, —CH(R²³)—CH(R²⁴)— or—C(R²³)═C(R²⁴)—; A¹ represents a single bond or C₁₋₂ alkylene; A²represents a single bond or —CH₂—; A³ represents C₁₋₃ alkylene; A⁴represents C(O) or C₁₋₂ alkylene; Z represents, at each occurrence, O,S(O)_(m) or N(R²⁵); m represents, at each occurrence, 0, 1 or 2; R² andR⁴ independently represent one or more optional substituents selectedfrom C₁₋₄ alkyl (which latter group is optionally substituted by one ormore halo substituent), C₁₋₄ alkoxy, methylenedioxy, halo, hydroxy,cyano, nitro, SO₂NH₂, C(O)OR²⁶ or N(R²⁷)R²⁸); R³ represents an optionalsubstituent selected from OH or C₁₋₄ alkoxy; R²¹, R²², R²³, R²⁴, R²⁵,R²⁶, R²⁷ and R²⁸ independently represent H or C₁₋₄ alkyl; Y representsCH₂, (CH₂)₂, CH═CH, (CH₂)₃, CH₂CH═CH or CH═CHCH₂, which latter threegroups are optionally substituted by C₁₋₄ alkyl, methylene, oxo orhydroxy; R^(y) represents H or C₁₋₄ alkyl; n represents 0, 1, 2, 3 or 4;and B represents a structural fragment of formula IIIa or IIIc

wherein X⁵, X⁶, X⁷ and X⁸ independently represent CH, N or N—O; and R³¹represents an optional substituent selected from halo and C₁₋₄ alkyl; ora pharmaceutically acceptable salt thereof; provided that: (a) A and Bdo not both represent O or S; (b) B and D do not both represent O or S;(c) when R¹ represents OR^(1d) and X₁ represents —C(O)-Z-A¹,-Z-CH₂—S(O)_(m)-A²-, —CH₂-Z-S(O)_(m)-A²- or -Z-C(O)-Z-A², then A¹ or A²(as appropriate) do not represent a single bond; and (d) when X₄represents —CH(R²³)—, R¹ does not represent OH.
 2. A compound of formulaI, as defined in claim 1, wherein R¹ represents OH or C₁₋₄ alkyl (whichlatter group is optionally substituted by cyano or OH).
 3. A compound offormula I, as defined in any one of the preceding claims, wherein R_(x)represents a structural fragment of formula IIa.
 4. A compound offormula I, as defined in any one of the preceding claims, wherein, whenR_(x) represents a structural fragment of formula IIa, the dotted linesrepresent bonds, A and B both represent CH and D represents —CH═CH—. 5.A compound of formula I, as defined in any one of the preceding claims,wherein, when R_(x) represents a structural fragment of formula IIa, X₁represents C₂- or C₃-alkylene, —O(CH₂)— or —O(CH₂)₂—.
 6. A compound offormula I, as defined in claim 5 wherein X₁ represents C₃-alkylene or—O(CH₂)₂—.
 7. A compound of formula I, as defined in any one of thepreceding claims, wherein Y represents CH₂, (CH₂)₂ or (CH₂)₃.
 8. Acompound of formula I, as defied in any one of the preceding claims,wherein, when B represents a structural fragment of formula IIIa, X⁵,X⁶, X⁷ and X⁸ all represents CH.
 9. A compound of formula I, as definedin any one of the preceding claims, wherein, when R_(x) represents astructural fragment of formula IIa, and R² represents at least onesubstituent, a point of substitution is at the carbon atom which is atposition B.
 10. A compound of formula I, as defined in any one of thepreceding claims, wherein, when R_(x) represents a structural fragmentof formula IIa, the dotted lines represent bonds, A and B both representCH, D represents —CH═CH—, and R² represents at least one substituent,the ring is substituted either at the carbon atom in the —CH═CH— group(position D) which is adjacent to the ring junction, or at the carbonatom which is at position B, or at both of these sites.
 11. A compoundof formula I, as defined in any one of the preceding claims, wherein thefragment

is in the S-configuration.
 12. A pharmaceutical formulation including acompound as defined in any one of claims 1 to 11, or a pharmaceuticallyacceptable salt thereof, in admixture with a pharmaceutically acceptableadjuvant, diluent or carrier.
 13. A compound as defined in any one ofclaims 1 to 11, or a pharmaceutically acceptable salt thereof, for useas a pharmaceutical.
 14. A compound as defined in any one of claims 1 to11, or a pharmaceutically acceptable salt thereof, for use in thetreatment of a condition where inhibition of thrombin is required.
 15. Acompound as defined in any one of claims 1 to 11, or a pharmaceuticallyacceptable salt thereof, for use in the treatment of thrombosis.
 16. Acompound as defined in any one of claims 1 to 11, or a pharmaceuticallyacceptable salt thereof, for use as an anticoagulant.
 17. The use of acompound as defined in any one of claims 1 to 11, or a pharmaceuticallyacceptable salt thereof, as active ingredient in the manufacture of amedicament for the treatment of a condition where inhibition of thrombinis required.
 18. The use as claimed in claim 17, wherein the conditionis thrombosis.
 19. The use of a compound as defined in any one of claims1 to 11, or a pharmaceutically acceptable salt thereof, as activeingredient in the manufacture of an anticoagulant.
 20. A method oftreatment of a condition where inhibition of thrombin is required whichmethod comprises administration of a therapeutically effective amount ofa compound as defined in any one of claims 1 to 11, or apharmaceutically acceptable salt thereof, to a person suffering from, orsusceptible to, such a condition.
 21. A method as claimed in claim 20,wherein the condition is thrombosis.
 22. A method as claimed in claim20, wherein the condition is hypercoagulability in blood and tissues.23. A process for the preparation of compounds of formula I whichcomprises: (a) a coupling reaction comprising: (i) the coupling of acompound of formula IV,

wherein R¹ and R_(x) are as defined in claim 1 with a compound offormula V,

wherein R^(y), Y, n and B are as defined in claim 1; or (ii) thecoupling of a compound of formula VI,

wherein R¹, R_(x) and Y are as defined in claim 1 with a compound offormula VII, H(R^(y))N—(CH₂)_(n)—B  VII wherein R^(y), n and B are asdefined in claim 1; (b) deprotection of a compound of formula Ia, asdefined in claim
 24. 24. A compound of formula Ia,

wherein B¹ represents a structural fragment of formula IIId or IIIf,

wherein D¹ and D² independently represent H, OH, OR^(a), OC(O)R^(b),OC(O)OR^(c), C(O)OR^(d), C(O)R^(e); in which R^(a) represents phenyl,benzyl, C₁₋₇ alkyl (which latter group is optionally interrupted byoxygen or is optionally substituted by halo) or—C(R^(f))(R^(g))—OC(O)R^(h); R^(b) represents C₁₋₁₇ alkyl (which lattergroup is optionally substituted by C₁₋₆ alkoxy, C₁₋₆ acyloxy, amino orhalo); C₁₋₆ alkoxy, C₃₋₇ cycloalkyl, phenyl, naphthyl or C₁₋₃alkylphenyl (which latter five groups are optionally substituted by C₁₋₆alkyl or halo); or —[C(R^(i))(R^(j))]_(m)OC(O)R^(k); R^(c) representsC₁₋₁₇ alkyl, phenyl, 2-naphthyl (which latter three groups areoptionally substituted by C₁₋₆ alkyl, Si(R^(aa))(R^(ab))(R^(ac)) orhalo), —[C(R^(m))(R^(n))]_(n)OC(O)R^(p), or —CH₂—Ar¹; R^(d) represents2-naphthyl, phenyl, C₁₋₃ alkylphenyl (which latter three groups areoptionally substituted by C₁₋₆ alkyl, C₁₋₆ alkoxy, nitro,Si(R^(ba))(R^(bb))(R^(bc)) or halo), C₁₋₁₂ alkyl (which latter group isoptionally substituted by C₁₋₆ alkoxy, C₁₋₆ acyloxy or halo),—[C(R^(q))(R^(r))]_(p)OC(O)R^(s) or —CH₂—AR²; R^(e) represents phenyl,benzyl, C₁₋₆ alkyl (which latter group is optionally interrupted byoxygen) or —[C(R^(t))(R^(u))]_(r)OC(O)R^(v); R^(aa), R^(ab), R^(ac),R^(ba), R^(bb) and R^(bc) independently represent C₁₋₆ alkyl or phenyl;R^(f), R^(g), R^(i), R^(j), R^(m), R^(n), R^(q), R^(r), R^(t) and R^(u)independently represent H or C₁₋₆ alkyl; R^(h), R^(k), R^(p), R^(s) andR^(v) independently represent C₁₋₁₇ alkyl (which latter group isoptionally substituted by C₁₋₆ alkoxy, C₁₋₆ acyloxy or halo); C₁₋₆alkoxy, C₃₋₇ cycloalkyl, phenyl, naphthyl or C₁₋₃ alkylphenyl (whichlatter five groups are optionally substituted by C₁₋₆ alkyl or halo);Ar¹ and Ar² independently represent the structural fragment

m and r independently represent 3 or 4; n and p independently represent1, 2 or 3; and R¹, R_(x), Y, R^(y), n, X⁵, X⁶, X⁷, X⁸ and R³¹ are ashereinbefore defined; or a pharmaceutically acceptable salt thereof;provided that D¹ and D² do not both represent H.
 25. A compound offormula Ia, as claimed in claim 24, wherein D¹ represents H and D²represents OH, OCH₃, OC(O)R^(b) or C(O)OR^(d), wherein R^(b) and R^(d)are as defined in claim 24.